Compositions comprising a GH61 polypeptide having cellulolytic enhancing activity and a liquor and method of using thereof

ABSTRACT

The present invention relates to compositions comprising: a polypeptide having cellulolytic enhancing activity and a liquor. The present invention also relates to methods of using the compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 15/175,645, filed Jun. 7, 2016, which is a continuation of U.S.patent application Ser. No. 13/814,912, filed Feb. 7, 2013, which is a35 U.S.C. § 371 national application of PCT/US2011/046795 filed Aug. 5,2011, which claims priority or the benefit under 35 U.S.C. 119 of U.S.Provisional Application Ser. No. 61/373,124, filed Aug. 12, 2010, U.S.Provisional Application Ser. No. 61/373,128, filed Aug. 12, 2010, U.S.Provisional Application Ser. No. 61/373,145, filed Aug. 12, 2010, U.S.Provisional Application Ser. No. 61/373,150, filed Aug. 12, 2010, U.S.Provisional Application Ser. No. 61/373,157, filed Aug. 12, 2010, U.S.Provisional Application Ser. No. 61/373,166, filed Aug. 12, 2010, U.S.Provisional Application Ser. No. 61/373,170, filed Aug. 12, 2010, andU.S. Provisional Application Ser. No. 61/373,210, filed Aug. 12, 2010,the contents of which are fully incorporated herein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under CooperativeAgreement DE-FC36-08GO18080 awarded by the Department of Energy. Thegovernment has certain rights in this invention.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form.The computer readable form is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to compositions comprising a polypeptidehaving cellulolytic enhancing activity and a liquor, and to methods ofusing the compositions.

Description of the Related Art

Cellulose is a polymer of the simple sugar glucose covalently linked bybeta-1,4-bonds. Many microorganisms produce enzymes that hydrolyzebeta-linked glucans. These enzymes include endoglucanases,cellobiohydrolases, and beta-glucosidases. Endoglucanases digest thecellulose polymer at random locations, opening it to attack bycellobiohydrolases. Cellobiohydrolases sequentially release molecules ofcellobiose from the ends of the cellulose polymer. Cellobiose is awater-soluble beta-1,4-linked dimer of glucose. Beta-glucosidaseshydrolyze cellobiose to glucose.

The conversion of lignocellulosic feedstocks into ethanol has theadvantages of the ready availability of large amounts of feedstock, thedesirability of avoiding burning or land filling the materials, and thecleanliness of the ethanol fuel. Wood, agricultural residues, herbaceouscrops, and municipal solid wastes have been considered as feedstocks forethanol production. These materials primarily consist of cellulose,hemicellulose, and lignin. Once the lignocellulose is converted tofermentable sugars, e.g., glucose, the fermentable sugars are easilyfermented by yeast into ethanol.

WO 2005/074647, WO 2008/148131, WO 2011/035027 disclose isolated GH61polypeptides having cellulolytic enhancing activity and thepolynucleotides thereof from Thielavia terrestris. WO 2005/074656 and WO2010/065830 disclose isolated GH61 polypeptides having cellulolyticenhancing activity and the polynucleotides thereof from Thermoascusaurantiacus. WO 2007/089290 discloses an isolated GH61 polypeptidehaving cellulolytic enhancing activity and the polynucleotide thereoffrom Trichoderma reesei. WO 2009/085935, WO 2009/085859, WO 2009/085864,and WO 2009/085868 disclose isolated GH61 polypeptides havingcellulolytic enhancing activity and the polynucleotides thereof fromMyceliophthora thermophila. WO 2010/138754 discloses isolated GH61polypeptides having cellulolytic enhancing activity and thepolynucleotides thereof from Aspergillus fumigatus. WO 2011/005867discloses isolated GH61 polypeptides having cellulolytic enhancingactivity and the polynucleotides thereof from Penicillium pinophilum. WO2011/039319 discloses isolated GH61 polypeptides having cellulolyticenhancing activity and the polynucleotides thereof from Thermoascus sp.WO 2011/041397 discloses isolated GH61 polypeptides having cellulolyticenhancing activity and the polynucleotides thereof from Penicillium sp.WO 2011/041504 discloses isolated GH61 polypeptides having cellulolyticenhancing activity and the polynucleotides thereof from Thermoascuscrustaceous. WO 2008/151043 discloses methods of increasing the activityof a GH61 polypeptide having cellulolytic enhancing activity by adding asoluble activating divalent metal cation to a composition comprising thepolypeptide.

It would be advantageous in the art to improve the ability ofpolypeptides having cellulolytic enhancing activity to enhance enzymatichydrolysis of lignocellulosic feedstocks.

The present invention relates to compositions comprising a polypeptidehaving cellulolytic enhancing activity and a liquor, and to methods ofusing the compositions.

SUMMARY OF THE INVENTION

The present invention relates to compositions comprising: (a) apolypeptide having cellulolytic enhancing activity; and (b) a liquor,wherein the combination of the polypeptide having cellulolytic enhancingactivity and the liquor enhances hydrolysis of a cellulosic material bya cellulolytic enzyme.

The present invention also relates to methods for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellulolytic enhancing activity and a liquor, wherein thecombination of the polypeptide having cellulolytic enhancing activityand the liquor enhances hydrolysis of the cellulosic material by theenzyme composition.

The present invention also relates to methods for producing afermentation product, comprising:

(a) saccharifying a cellulosic material with an enzyme composition inthe presence of a polypeptide having cellulolytic enhancing activity anda liquor, wherein the combination of the polypeptide having cellulolyticenhancing activity and the liquor enhances hydrolysis of the cellulosicmaterial by the enzyme composition;

(b) fermenting the saccharified cellulosic material with one or more(e.g., several) fermenting microorganisms to produce the fermentationproduct; and

(c) recovering the fermentation product from the fermentation.

The present invention also relates to methods of fermenting a cellulosicmaterial, comprising: fermenting the cellulosic material with one ormore (e.g., several) fermenting microorganisms, wherein the cellulosicmaterial is saccharified with an enzyme composition in the presence of apolypeptide having cellulolytic enhancing activity and a liquor, whereinthe combination of the polypeptide having cellulolytic enhancingactivity and the liquor enhances hydrolysis of the cellulosic materialby the enzyme composition.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the fractional hydrolysis of washed and unwashed pretreatedcorn stover by a Trichoderma reesei cellulase composition with variousconcentrations of Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancement activity. Open symbols: 1 day of hydrolysis;closed symbols: 3 days of hydrolysis; squares: milled, water-washedpretreated corn stover; circles: milled, unwashed pretreated cornstover; triangles: hot-water washed, milled pretreated corn stover.

FIGS. 2A and 2B show the effect of acid-pretreated corn stover liquor onGH61 polypeptide-enhancement of cellulolysis of milled, water-washedpretreated corn stover by a T. reesei cellulase composition. FIG. 2A:fractional hydrolysis. White bars: 1 day hydrolysis; gray bars: 3 dayhydrolysis. FIG. 2B: open symbols, 1 day hydrolysis; solid symbols: 3day hydrolysis. Circles: no added liquor; squares: 2% (v/v) liquor;diamonds: 5% (v/v) liquor; triangles: 10% liquor; inverted triangles:15% liquor. Data were fit linearly or by a modified saturation-bindingmodel as described.

FIGS. 3A and 3B show acid-pretreated corn stover liquor dependence of T.aurantiacus GH61A polypeptide-enhancement of hydrolysis of pretreatedcorn stover. FIG. 3A: AVICEL®+various concentrations of theacid-pretreated corn stover liquor as a function of GH61 polypeptideconcentrations. FIG. 3B: AVICEL®+synthetic liquor as a function of GH61polypeptide concentration. Open symbols: 1 day hydrolysis; closedsymbols: 3 days hydrolysis. Circles: no liquor; diamonds: 5% (v/v)liquor; triangles: 10% liquor; inverted triangles: 15% liquor; squares:5% synthetic liquor containing no phenol; right triangles: 15% syntheticliquor containing no phenol. Data were fit linearly or using Equation 2,as described.

FIGS. 4A, 4B, and 4C show the effect of enzymatically treated or notenzymatically-treated pretreated corn stover liquors on enhancement ofcellulolysis of pretreated corn stover by the Thelavia terrestris GH61Epolypeptide. FIG. 4A: un-treated liquor; FIG. 4B: T. reeseicellulase-treated liquor; and FIG. 4C: T. reesei cellulase and Thelaviaterrestris GH61E polypeptide-treated liquor. Circles: no added liquor;squares: 5% (v/v) liquor; diamonds: 10% (v/v) liquor; triangles: 15%(v/v) liquor. Data were fit linearly or using Equation 2, as described.

FIGS. 5A and 5B show the fractional hydrolysis of microcrystallinecellulose by the T. reesei cellulase composition with various T.aurantiacus GH61A polypeptide concentrations, comparing impact ofaddition of dilute-acid and steam pretreatment liquors at 5 days ofhydrolysis. FIG. 5A: NREL acid-pretreated corn stover liquor; FIG. 5B:steam explosion-pretreated corn stover liquor. Open symbols: 5% (v/v)liquor; closed symbols: 15% (v/v) liquor. Circles: whole liquor;squares: low molecular weight fraction, triangles: high molecular weightfraction. Data were fit using Equation 2, as described.

FIG. 6 shows the effect of retentates and flow-through samples ofmolecular weight-filtered acid-pretreated corn stover liquor on GH61polypeptide cellulolytic enhancing activity. White bars: 1 day ofsaccharification; gray bars: 6 days of saccharification. Concentrationslisted refer to the T. reesei cellulase composition and the GH61polypeptide concentration, respectively, in mg per gram cellulose.

FIG. 7A shows the fractional hydrolysis of microcrystalline cellulose byindividual T. reesei cellulase monocomponents and mixtures thereof, andthe effects of the T. aurantiacus GH61A polypeptide and NRELacid-pretreated corn stover liquor thereon. FIG. 7B shows the GH61effect on the individual cellulases and mixtures of cellulases. Whitebars: 3 days of hydrolysis; gray bars: 5 days of hydrolysis; black bars:7 days of hydrolysis.

FIG. 8A shows HPLC chromatography of NREL acid-pretreated corn stoverliquor. Fractional hydrolysis and absorbance are plotted for the variousHPLC fractions. Solid line: fractional hydrolysis; light dashed line:absorbance at 210 nm; heavy dashed line: absorbance at 280 nm. Theaverage hydrolysis for all samples is indicated by the solid, horizontalline. FIG. 8B shows the fractional hydrolysis for the T. reeseicellulase composition with increasing GH61A polypeptide concentrationsin the presence of 3 kDa MWCO flow-through fractions of NRELacid-pretreated corn stover liquor incubated with microcrystallinecellulose and eluted with successive water and organic solvent washes.Gray bars: 3 days of hydrolysis; black bars: 7 days of hydrolysis.

FIG. 9 shows a standard curve of AVICEL® height vs. added mass.

FIG. 10 shows the effect of various pooled, HPLC-separated NRELacid-pretreated corn stover liquor fractions and the T. aurantiacusGH61A polypeptide on hydrolysis of microcrystalline cellulose by the T.reesei cellulase composition. Solid lines: height of AVICEL® incubatedwith the GH61 polypeptide; dashed lines: A_((600nm)) of washed, BCAreagent-reacted AVICEL®.

FIG. 11A shows a standard curve of reducing sugar equivalents and FIG.11B shows the reducing sugar equivalents measured in the solidmicrocrystalline cellulose incubated with the T. aurantiacus GH61Apolypeptide and the indicated HPLC fractions.

FIG. 12 shows the results of a microcrystalline cellulose hydrolysisassay with the T. reesei cellulase composition in the presence of the T.aurantiacus GH61A polypeptide and chromatographed fractions ofacid-pretreated xylan. Solid line: fractional hydrolysis with GH61;dashed line: mean fractional hydrolysis.

FIG. 13 shows a LC-MS chromatogram of a representative HPLC fraction ofacid-pretreated xylan.

FIGS. 14A, 14B, 14C, and 14D shows liquid chromatography-massspectrometry chromatograms of GH61 polypeptide-affinity enrichedacid-pretreated corn stover. FIG. 14A: diode array detection; FIG. 14B:TOF MS/MS total ion current 17.5; FIG. 14C: TOF MS/MS ES-total ioncurrent 273; FIG. 14D: TOF MS ES-total ion survey.

FIGS. 15A and 15B shows ion chromatograms of microcrystalline celluloseor phosphoric acid-swollen cellulose incubated with the T. aurantiacusGH61A polypeptide and NREL acid-pretreated corn stover liquor. FIG. 15A:reaction samples; FIG. 15B: comparison to analytical standards. FIG.15A: solid lines: AVICEL® incubations; dashed lines: PASO incubations;light gray: AVICEL®; medium gray: AVICEL®+NREL acid-pretreated cornstover liquor; dark gray: AVICEL®+GH61 polypeptide; black: AVICEL®+GH61polypeptide+liquor. FIG. 15B: black: AVICEL®+GH61 polypeptide+liquor;dark gray solid lines: liquor; dark gray dashed lines: cellopentaose,cellotetraose and cellotriose; light gray: cellopentaonic acid,cellotetraonic acid, gluconic acid, galactonic acid and xylotetraose,peaks as indicated.

FIG. 16A shows the fractional hydrolysis of AVICEL® by a T. reeseicellulase composition with the indicated T. aurantiacus GH61Apolypeptide concentration in the presence of alkaline-pretreated cornstover generated using the indicated pretreatment concentration ofsodium hydroxide. White bars: 1 day of hydrolysis; gray bars: 3 days ofhydrolysis; black bars: 7 days of hydrolysis. FIG. 16B shows thefractional hydrolysis of AVICEL® by the T. reesei cellulase compositionwith the indicated GH61 polypeptide concentration in the presence ofwater-extracted, acid-pretreated corn stover liquors generated using theindicated extraction temperature. Gray bars: 1 day of hydrolysis; whitebars: 3 days of hydrolysis.

FIGS. 17A and 17B show the fractional hydrolysis of AVICEL® by a T.reesei cellulase composition with various concentrations of the T.aurantiacus GH61A polypeptide in the presence of acid-pretreatedcomponents of biomass or mixtures thereof. White bars: 1 day ofhydrolysis; dark gray bars: 5 days of hydrolysis.

FIG. 18 shows the fractional hydrolysis of AVICEL® by a T. reeseicellulase composition with various concentrations of the T. aurantiacusGH61A polypeptide in the presence of post-fermentation liquors. Whitebars: 1 day of hydrolysis; gray bars: 3 days of hydrolysis; black bars:7 days of hydrolysis.

FIGS. 19A and 19B show the fractional hydrolysis of AVICEL® by a T.reesei cellulase composition with various concentrations of the T.aurantiacus GH61A polypeptide in the presence of various severityacid-pretreatments of corn stover. Gray bars: 3 days of hydrolysis;black bars: 7 days of hydrolysis. FIG. 19C shows the fractionalhydrolysis of AVICEL® by the T. reesei cellulase composition with T.aurantiacus GH61A polypeptide and liquors generated by acid pretreatmentof cellulose at various severities between 110° C. and 190° C. at GH61Apolypeptide concentrations of 50% ( ), 24% ( ); 8% ( ); 4% ( ); 2% ( );and 0 ( ) (w/w).

FIGS. 20A, 20B, and 20C shows the fractional hydrolysis of AVICEL® by T.reesei cellulase compositions with increasing GH61 polypeptideconcentrations plus added acid-pretreated xylan of various severities.FIG. 20A shows 7 days of hydrolysis data for a broad range ofseverities. FIG. 20B shows more severe pretreatments, gray bars: 3 daysof hydrolysis; black bars: 7 days of hydrolysis. FIG. 20C shows thefractional hydrolysis of AVICEL® by the T. reesei cellulase compositionwith T. aurantiacus GH61A polypeptide and liquors generated by acidpretreatment of xylan at various severities between 110° C. and 190° C.at GH61A polypeptide concentrations of 50% ( ), 24% ( ); 8% ( ); 4% ( );2% ( ); and 0 ( ) (w/w).

FIG. 21 shows the fractional hydrolysis of AVICEL® by T. reeseicellulase compositions with and without 50% (w/w) T. aurantiacus GH61Apolypeptide concentrations plus added solid-phase extracted NRELacid-pretreated corn stover or acid-pretreated xylan as indicated. Graybars: 3 days of hydrolysis; black bars: 7 days of hydrolysis.

FIG. 22 shows the fractional hydrolysis of AVICEL® by a T. reeseicellulase composition with various concentrations of T. aurantiacusGH61A in the presence of 10% (v/v) of the indicated pretreatment liquoror electrodialyzed pretreatment liquor. Gray bars: 3 days of hydrolysis;black bars: 7 days of hydrolysis.

FIGS. 23A, 23B, 23C, and 23D show the fractional hydrolysis of variousbiomass substrates by the T. reesei cellulase composition withincreasing concentrations of the T. aurantiacus GH61A polypeptide withand without NREL acid-pretreated corn stover liquor. FIG. 23A: low andmedium severity organosolv ethanol pretreated corn stover; FIG. 23B:medium severity glycerol and water pretreated corn stover, and 5% totalsolids water pretreated corn stover; FIG. 23C: 5% total solids sugarcanebagasse; FIG. 23D: alkaline pretreated corn stover plus no liquor andNREL milled washed pretreated corn stover controls. White bars: 1 day ofhydrolysis; gray bars: 3 days of hydrolysis; black bars: 7 days ofhydrolysis.

FIG. 24 shows the fractional hydrolysis of AVICEL® by the T. reeseicellulase composition with either zero or 24% T. aurantiacus GH61Apolypeptide in the presence of the indicated acid-pretreatedmonosaccharides. Gray bars: 3 days of hydrolysis; black bars: 7 days ofhydrolysis.

FIG. 25 shows the fractional hydrolysis of AVICEL® by the T. reeseicellulase composition with various concentrations of the indicated GH61polypeptides with 10% (v/v) NREL PCS liquor. White symbols: 3 days ofhydrolysis; black symbols: 7 days of hydrolysis; Thermoascus aurantiacusGH61A: circles; Aspergillus fumigatus GH61B polypeptide: diamonds;Penicillium pinophilum GH61 polypeptide: squares.

FIG. 26 shows the glucose produced by hydrolysis of AVICEL® by a T.reesei cellulase composition with T. aurantiacus GH61A polypeptide inthe presence or absence of Kraft lignin. Solid symbols: T. reeseicellulase composition+T. aurantiacus GH61A polypeptide. Open symbols: T.reesei cellulase composition+T. aurantiacus GH61A polypeptidesupplemented with additional 15% (w/w) T. aurantiacus GH61A polypeptide.Circles: no lignin; squares: 0.1% (w/w) Kraft lignin; diamonds: 0.1%(w/w) oxidized Kraft lignin.

FIG. 27 shows the concentrations of glucose and xylose from 120 hours ofsaccharification of washed, milled alkaline pretreated corn stover bythe T. reesei cellulase composition supplemented with T. aurantiacusGH61A polypeptide, replaced with increasing concentrations of T.aurantiacus GH61A. Solid squares: xylose, open diamonds: glucose.

FIG. 28 shows the conversion of high total solids (15% TS) dilute acidpretreated corn stover of various pretreatment severities as indicated.The pretreated corn stovers were hydrolyzed by either a compositioncontaining a blend of an Aspergillus aculeatus GH10 xylanase and aTrichoderma reesei cellulase preparation containing Aspergillusfumigatus beta-glucosidase and Thermoascus aurantiacus GH61A polypeptideor this mixture replaced with 20% additional T. aurantiacus GH61A. Foreach severity pretreatment other than the least severe, replacement ofthe cellulase-GH61A polypeptide mixture with additional GH61Apolypeptide yielded a greater conversion. Grey bars: 120 hours ofsaccharification; black bars: 216 hours of saccharification.

FIG. 29 shows the conversion of high total solids (15% TS) dilute acidpretreated Arundo donax of various pretreatment severities as indicated.The variously pretreated A. donax were hydrolyzed by either acomposition containing a blend of an Aspergillus aculeatus GH10 xylanase(WO 94/021785) and a Trichoderma reesei cellulase preparation containingAspergillus fumigatus beta-glucosidase (WO 2005/047499) and Thermoascusaurantiacus GH61A polypeptide (WO 2005/074656) or this mixture replacedby 20% additional T. aurantiacus GH61A polypeptide. For each severitypretreatment, replacement of the cellulase-GH61A polypeptide mixturewith additional GH61A polypeptide yielded a greater conversion. Greybars: 120 hours of saccharification; black bars: 216 hours ofsaccharification.

DEFINITIONS

Acetylxylan esterase: The term “acetylxylan esterase” means acarboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of acetylgroups from polymeric xylan, acetylated xylose, acetylated glucose,alpha-napthyl acetate, and p-nitrophenyl acetate. For purposes of thepresent invention, acetylxylan esterase activity is determined using 0.5mM p-nitrophenylacetate as substrate in 50 mM sodium acetate pH 5.0containing 0.01% TWEEN™ 20 (polyoxyethylene sorbitan monolaurate). Oneunit of acetylxylan esterase is defined as the amount of enzyme capableof releasing 1 μmole of p-nitrophenolate anion per minute at pH 5, 25°C.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Alpha-L-arabinofuranosidase: The term “alpha-L-arabinofuranosidase”means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55)that catalyzes the hydrolysis of terminal non-reducingalpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzymeacts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)-and/or (1,5)-linkages, arabinoxylans, and arabinogalactans.Alpha-L-arabinofuranosidase is also known as arabinosidase,alpha-arabinosidase, alpha-L-arabinosidase, alpha-arabinofuranosidase,polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranosidehydrolase, L-arabinosidase, or alpha-L-arabinanase. For purposes of thepresent invention, alpha-L-arabinofuranosidase activity is determinedusing 5 mg of medium viscosity wheat arabinoxylan (MegazymeInternational Ireland, Ltd., Bray, Co. Wicklow, Ireland) per ml of 100mM sodium acetate pH 5 in a total volume of 200 μl for 30 minutes at 40°C. followed by arabinose analysis by AMINEX® HPX-87H columnchromatography (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

Alpha-glucuronidase: The term “alpha-glucuronidase” means analpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139) that catalyzesthe hydrolysis of an alpha-D-glucuronoside to D-glucuronate and analcohol. For purposes of the present invention, alpha-glucuronidaseactivity is determined according to de Vries, 1998, J. Bacteriol. 180:243-249. One unit of alpha-glucuronidase equals the amount of enzymecapable of releasing 1 μmole of glucuronic or 4-O-methylglucuronic acidper minute at pH 5, 40° C.

Beta-glucosidase: The term “beta-glucosidase” means a beta-D-glucosideglucohydrolase (E.C. 3.2.1.21) that catalyzes the hydrolysis of terminalnon-reducing beta-D-glucose residues with the release of beta-D-glucose.For purposes of the present invention, beta-glucosidase activity isdetermined using p-nitrophenyl-beta-D-glucopyranoside as substrateaccording to the procedure of Venturi et al., 2002, Extracellularbeta-D-glucosidase from Chaetomium thermophilum var. coprophilum:production, purification and some biochemical properties, J. BasicMicrobiol. 42: 55-66. One unit of beta-glucosidase is defined as 1.0μmole of p-nitrophenolate anion produced per minute at 25° C., pH 4.8from 1 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mMsodium citrate containing 0.01% TWEEN® 20.

Beta-xylosidase: The term “beta-xylosidase” means a beta-D-xylosidexylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of shortbeta (1→4)-xylooligosaccharides, to remove successive D-xylose residuesfrom non-reducing termini. For purposes of the present invention, oneunit of beta-xylosidase is defined as 1.0 μmole of p-nitrophenolateanion produced per minute at 40° C., pH 5 from 1 mMp-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodium citratecontaining 0.01% TWEEN® 20.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic cell. cDNA lacks intron sequences that may be presentin the corresponding genomic DNA. The initial, primary RNA transcript isa precursor to mRNA that is processed through a series of steps,including splicing, before appearing as mature spliced mRNA.

Cellobiohydrolase: The term “cellobiohydrolase” means a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91) that catalyzes thehydrolysis of 1,4-beta-D-glucosidic linkages in cellulose,cellooligosaccharides, or any beta-1,4-linked glucose containingpolymer, releasing cellobiose from the reducing or non-reducing ends ofthe chain (Teeri, 1997, Crystalline cellulose degradation: New insightinto the function of cellobiohydrolases, Trends in Biotechnology 15:160-167; Teeri et al., 1998, Trichoderma reesei cellobiohydrolases: whyso efficient on crystalline cellulose?, Biochem. Soc. Trans. 26:173-178). For purposes of the present invention, cellobiohydrolaseactivity is determined according to the procedures described by Lever etal., 1972, Anal. Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBSLetters, 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters,187: 283-288; and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581. Inthe present invention, the Lever et al. method can be employed to assesshydrolysis of cellulose in corn stover, while the methods of vanTilbeurgh et al. and Tomme et al. can be used to determine thecellobiohydrolase activity on a fluorescent disaccharide derivative,4-methylumbelliferyl-β-D-lactoside.

Cellulolytic enhancing activity: The term “cellulolytic enhancingactivity” means a biological activity catalyzed by a GH61 polypeptidethat enhances the hydrolysis of a cellulosic material by enzyme havingcellulolytic activity. For purposes of the present invention,cellulolytic enhancing activity is determined by measuring the increasein reducing sugars or the increase of the total of cellobiose andglucose from the hydrolysis of a cellulosic material by cellulolyticenzyme under the following conditions: 1-50 mg of total protein/g ofcellulose in PCS, wherein total protein is comprised of 50-99.5% w/wcellulolytic enzyme protein and 0.5-50% w/w protein of a GH61polypeptide having cellulolytic enhancing activity for 1-7 days at 50°C. compared to a control hydrolysis with equal total protein loadingwithout cellulolytic enhancing activity (1-50 mg of cellulolyticprotein/g of cellulose in PCS). In a preferred aspect, a mixture ofCELLUCLAST® 1.5 L (Novozymes A/S, Bagsvrd, Denmark) in the presence of2-3% of total protein weight Aspergillus oryzae beta-glucosidase(recombinantly produced in Aspergillus oryzae according to WO 02/095014)or 2-3% of total protein weight Aspergillus fumigatus beta-glucosidase(recombinantly produced in Aspergillus oryzae as described in WO2002/095014) of cellulase protein loading is used as the source of thecellulolytic activity.

The GH61 polypeptides having cellulolytic enhancing activity enhance thehydrolysis of a cellulosic material catalyzed by enzyme havingcellulolytic activity by reducing the amount of cellulolytic enzymerequired to reach the same degree of hydrolysis preferably at least1.01-fold, more preferably at least 1.05-fold, more preferably at least1.10-fold, more preferably at least 1.25-fold, more preferably at least1.5-fold, more preferably at least 2-fold, more preferably at least3-fold, more preferably at least 4-fold, more preferably at least5-fold, even more preferably at least 10-fold, and most preferably atleast 20-fold.

Cellulolytic enzyme or cellulase: The term “cellulolytic enzyme” or“cellulase” means one or more (e.g., several) enzymes that hydrolyze acellulosic material. Such enzymes include endoglucanase(s),cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. Thetwo basic approaches for measuring cellulolytic activity include: (1)measuring the total cellulolytic activity, and (2) measuring theindividual cellulolytic activities (endoglucanases, cellobiohydrolases,and beta-glucosidases) as reviewed in Zhang et al., Outlook forcellulase improvement: Screening and selection strategies, 2006,Biotechnology Advances 24: 452-481. Total cellulolytic activity isusually measured using insoluble substrates, including Whatman No 1filter paper, microcrystalline cellulose, bacterial cellulose, algalcellulose, cotton, pretreated lignocellulose, etc. The most common totalcellulolytic activity assay is the filter paper assay using Whatman No.1 filter paper as the substrate. The assay was established by theInternational Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987,Measurement of cellulase activities, Pure Appl. Chem. 59: 257-68).

For purposes of the present invention, cellulolytic enzyme activity isdetermined by measuring the increase in hydrolysis of a cellulosicmaterial by cellulolytic enzyme(s) under the following conditions: 1-20mg of cellulolytic enzyme protein/g of cellulose in PCS for 3-7 days at50° C. compared to a control hydrolysis without addition of cellulolyticenzyme protein. Typical conditions are 1 ml reactions, washed orunwashed PCS, 5% insoluble solids, 50 mM sodium acetate pH 5, 1 mMMnSO₄, 50° C., 72 hours, sugar analysis by AMINEX® HPX-87H column(Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

Cellulosic material: The term “cellulosic material” means any materialcontaining cellulose. The predominant polysaccharide in the primary cellwall of biomass is cellulose, the second most abundant is hemicellulose,and the third is pectin. The secondary cell wall, produced after thecell has stopped growing, also contains polysaccharides and isstrengthened by polymeric lignin covalently cross-linked tohemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thusa linear beta-(1-4)-D-glucan, while hemicelluloses include a variety ofcompounds, such as xylans, xyloglucans, arabinoxylans, and mannans incomplex branched structures with a spectrum of substituents. Althoughgenerally polymorphous, cellulose is found in plant tissue primarily asan insoluble crystalline matrix of parallel glucan chains.Hemicelluloses usually hydrogen bond to cellulose, as well as to otherhemicelluloses, which help stabilize the cell wall matrix.

Cellulose is generally found, for example, in the stems, leaves, hulls,husks, and cobs of plants or leaves, branches, and wood of trees. Thecellulosic material can be, but is not limited to, agricultural residue,herbaceous material (including energy crops), municipal solid waste,pulp and paper mill residue, waste paper, and wood (including forestryresidue) (see, for example, Wiselogel et al., 1995, in Handbook onBioethanol (Charles E. Wyman, editor), pp. 105-118, Taylor & Francis,Washington D.C.; Wyman, 1994, Bioresource Technology 50: 3-16; Lynd,1990, Applied Biochemistry and Biotechnology 24/25: 695-719; Mosier etal., 1999, Recent Progress in Bioconversion of Lignocellulosics, inAdvances in Biochemical Engineering/Biotechnology, T. Scheper, managingeditor, Volume 65, pp.23-40, Springer-Verlag, New York). It isunderstood herein that the cellulose may be in the form oflignocellulose, a plant cell wall material containing lignin, cellulose,and hemicellulose in a mixed matrix. In a preferred aspect, thecellulosic material is any biomass material. In another preferredaspect, the cellulosic material is lignocellulose, which comprisescellulose, hemicelluloses, and lignin.

In one aspect, the cellulosic material is agricultural residue. Inanother aspect, the cellulosic material is herbaceous material(including energy crops). In another aspect, the cellulosic material ismunicipal solid waste. In another aspect, the cellulosic material ispulp and paper mill residue. In another aspect, the cellulosic materialis waste paper. In another aspect, the cellulosic material is wood(including forestry residue).

In another aspect, the cellulosic material is arundo. In another aspect,the cellulosic material is bagasse. In another aspect, the cellulosicmaterial is bamboo. In another aspect, the cellulosic material is corncob. In another aspect, the cellulosic material is corn fiber. Inanother aspect, the cellulosic material is corn stover. In anotheraspect, the cellulosic material is miscanthus. In another aspect, thecellulosic material is orange peel. In another aspect, the cellulosicmaterial is rice straw. In another aspect, the cellulosic material isswitchgrass. In another aspect, the cellulosic material is wheat straw.

In another aspect, the cellulosic material is aspen. In another aspect,the cellulosic material is eucalyptus. In another aspect, the cellulosicmaterial is fir. In another aspect, the cellulosic material is pine. Inanother aspect, the cellulosic material is poplar. In another aspect,the cellulosic material is spruce. In another aspect, the cellulosicmaterial is willow.

In another aspect, the cellulosic material is algal cellulose. Inanother aspect, the cellulosic material is bacterial cellulose. Inanother aspect, the cellulosic material is cotton linter. In anotheraspect, the cellulosic material is filter paper. In another aspect, thecellulosic material is microcrystalline cellulose. In another aspect,the cellulosic material is phosphoric-acid treated cellulose.

In another aspect, the cellulosic material is an aquatic biomass. Asused herein the term “aquatic biomass” means biomass produced in anaquatic environment by a photosynthesis process. The aquatic biomass canbe algae, emergent plants, floating-leaf plants, or submerged plants.

The cellulosic material may be used as is or may be subjected topretreatment, using conventional methods known in the art, as describedherein. In a preferred aspect, the cellulosic material is pretreated.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which usually begins with the ATG start codon oralternative start codons such as GTG and TTG and ends with a stop codonsuch as TAA, TAG, and TGA. The coding sequence may be a DNA, cDNA,synthetic, or recombinant polynucleotide.

Control sequences: The term “control sequences” means all componentsnecessary for the expression of a polynucleotide encoding a polypeptide.Each control sequence may be native or foreign to the polynucleotideencoding the polypeptide or native or foreign to each other. Suchcontrol sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the polynucleotideencoding a polypeptide.

Endoglucanase: The term “endoglucanase” means anendo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4) thatcatalyzes endohydrolysis of 1,4-beta-D-glycosidic linkages in cellulose,cellulose derivatives (such as carboxymethyl cellulose and hydroxyethylcellulose), lichenin, beta-1,4 bonds in mixed beta-1,3 glucans such ascereal beta-D-glucans or xyloglucans, and other plant materialcontaining cellulosic components. Endoglucanase activity can bedetermined by measuring reduction in substrate viscosity or increase inreducing ends determined by a reducing sugar assay (Zhang et al., 2006,Biotechnology Advances 24: 452-481). For purposes of the presentinvention, endoglucanase activity is determined using carboxymethylcellulose (CMC) as substrate according to the procedure of Ghose, 1987,Pure and Appl. Chem. 59: 257-268, at pH 5, 40° C.

Expression: The term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to additional nucleotides thatprovide for its expression.

Family 61 glycoside hydrolase: The term “Family 61 glycoside hydrolase”or “Family GH61” or “GH61” means a polypeptide falling into theglycoside hydrolase Family 61 according to Henrissat B., 1991, Aclassification of glycosyl hydrolases based on amino-acid sequencesimilarities, Biochem. J. 280: 309-316, and Henrissat B., and BairochA., 1996, Updating the sequence-based classification of glycosylhydrolases, Biochem. J. 316: 695-696. The enzymes in this family wereoriginally classified as a glycoside hydrolase family based onmeasurement of very weak endo-1,4-beta-D-glucanase activity in onefamily member. The structure and mode of action of these enzymes arenon-canonical and they cannot be considered as bona fide glycosidases.However, they are kept in the CAZy classification on the basis of theircapacity to enhance the breakdown of lignocellulose when used inconjunction with a cellulase or a mixture of cellulases.

Feruloyl esterase: The term “feruloyl esterase” means a4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) thatcatalyzes the hydrolysis of 4-hydroxy-3-methoxycinnamoyl (feruloyl)groups from esterified sugar, which is usually arabinose in “natural”substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate). Feruloylesterase is also known as ferulic acid esterase, hydroxycinnamoylesterase, FAE-III, cinnamoyl ester hydrolase, FAEA, cinnAE, FAE-I, orFAE-II. For purposes of the present invention, feruloyl esteraseactivity is determined using 0.5 mM p-nitrophenylferulate as substratein 50 mM sodium acetate pH 5.0. One unit of feruloyl esterase equals theamount of enzyme capable of releasing 1 μmole of p-nitrophenolate anionper minute at pH 5, 25° C.

Hemicellulolytic enzyme or hemicellulase: The term “hemicellulolyticenzyme” or “hemicellulase” means one or more (e.g., several) enzymesthat hydrolyze a hemicellulosic material. See, for example, Shallom, D.and Shoham, Y. Microbial hemicellulases. Current Opinion InMicrobiology, 2003, 6(3): 219-228). Hemicellulases are key components inthe degradation of plant biomass. Examples of hemicellulases include,but are not limited to, an acetylmannan esterase, an acetyxylanesterase, an arabinanase, an arabinofuranosidase, a coumaric acidesterase, a feruloyl esterase, a galactosidase, a glucuronidase, aglucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and axylosidase. The substrates of these enzymes, the hemicelluloses, are aheterogeneous group of branched and linear polysaccharides that arebound via hydrogen bonds to the cellulose microfibrils in the plant cellwall, crosslinking them into a robust network. Hemicelluloses are alsocovalently attached to lignin, forming together with cellulose a highlycomplex structure. The variable structure and organization ofhemicelluloses require the concerted action of many enzymes for itscomplete degradation. The catalytic modules of hemicellulases are eitherglycoside hydrolases (GHs) that hydrolyze glycosidic bonds, orcarbohydrate esterases (CEs), which hydrolyze ester linkages of acetateor ferulic acid side groups. These catalytic modules, based on homologyof their primary sequence, can be assigned into GH and CE familiesmarked by numbers. Some families, with an overall similar fold, can befurther grouped into clans, marked alphabetically (e.g., GH-A). A mostinformative and updated classification of these and other carbohydrateactive enzymes is available in the Carbohydrate-Active Enzymes (CAZy)database. Hemicellulolytic enzyme activities can be measured accordingto Ghose and Bisaria, 1987, Pure & Appl. Chem. 59: 1739-1752.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, and the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Isolated or Purified: The term “isolated” or “purified” means apolypeptide or polynucleotide that is removed from at least onecomponent with which it is naturally associated. For example, apolypeptide may be at least 1% pure, e.g., at least 5% pure, at least10% pure, at least 20% pure, at least 40% pure, at least 60% pure, atleast 80% pure, at least 90% pure, or at least 95% pure, as determinedby SDS-PAGE, and a polynucleotide may be at least 1% pure, e.g., atleast 5% pure, at least 10% pure, at least 20% pure, at least 40% pure,at least 60% pure, at least 80% pure, at least 90% pure, or at least 95%pure, as determined by agarose electrophoresis.

Liquor: The term “liquor” means the solution phase, either aqueous,organic, or a combination thereof, arising from treatment of alignocellulose and/or hemicellulose and/or lignacious material orfeedstock, or monosaccharides thereof, e.g., xylose, arabinose, mannose,etc., under conditions as described herein, and the soluble contentsthereof.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. It is known in the art that a hostcell may produce a mixture of two of more different mature polypeptides(i.e., with a different C-terminal and/or N-terminal amino acid)expressed by the same polynucleotide. The mature polypeptide can bepredicted using the SignalP program (Nielsen et al., 1997, ProteinEngineering 10: 1-6).

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” is defined herein as a nucleotide sequence that encodes amature polypeptide having biological activity. The mature polypeptidecoding sequence can be predicted using the SignalP program (Nielsen etal., 1997, supra).

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic. The term nucleic acid construct issynonymous with the term “expression cassette” when the nucleic acidconstruct contains the control sequences required for expression of acoding sequence of the present invention.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs the expression of the coding sequence.

Polypeptide fragment: The term “fragment” means a polypeptide having oneor more (e.g., several) amino acids deleted from the amino and/orcarboxyl terminus of a mature polypeptide; wherein the fragment hasbiological activity.

Pretreated corn stover: The term “PCS” or “Pretreated Corn Stover” meansa cellulosic material derived from corn stover by treatment with heatand dilute sulfuric acid, alkaline pretreatment, or neutralpretreatment.

Sequence Identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the degree of sequence identitybetween two amino acid sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0, 5.0.0,or later. The optional parameters used are gap open penalty of 10, gapextension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the -nobrief option) is used as the percent identity andis calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the degree of sequence identitybetween two deoxyribonucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,supra), preferably version 3.0.0 or later. The optional parameters usedare gap open penalty of 10, gap extension penalty of 0.5, and theEDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the -nobriefoption) is used as the percent identity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides deleted from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having biological activity.

Variant: The term “variant” means a polypeptide having cellulolyticenhancing activity comprising an alteration, i.e., a substitution,insertion, and/or deletion of one or more (e.g., several) amino acidresidues at one or more (e.g., several) positions. A substitution meansa replacement of an amino acid occupying a position with a differentamino acid; a deletion means removal of an amino acid occupying aposition; and an insertion means adding one or more (e.g., several)amino acids, e.g., 1-5 amino acids, adjacent to an amino acid occupyinga position.

Xylan-containing material: The term “xylan-containing material” meansany material comprising a plant cell wall polysaccharide containing abackbone of beta-(1-4)-linked xylose residues. Xylans of terrestrialplants are heteropolymers possessing a beta-(1-4)-D-xylopyranosebackbone, which is branched by short carbohydrate chains. They compriseD-glucuronic acid or its 4-O-methyl ether, L-arabinose, and/or variousoligosaccharides, composed of D-xylose, L-arabinose, D- or L-galactose,and D-glucose. Xylan-type polysaccharides can be divided into homoxylansand heteroxylans, which include glucuronoxylans,(arabino)glucuronoxylans, (glucurono)arabinoxylans, arabinoxylans, andcomplex heteroxylans. See, for example, Ebringerova et al., 2005, Adv.Polym. Sci. 186: 1-67.

In the methods of the present invention, any material containing xylanmay be used. In a preferred aspect, the xylan-containing material islignocellulose.

Xylan degrading activity or xylanolytic activity: The term “xylandegrading activity” or “xylanolytic activity” means a biologicalactivity that hydrolyzes xylan-containing material. The two basicapproaches for measuring xylanolytic activity include: (1) measuring thetotal xylanolytic activity, and (2) measuring the individual xylanolyticactivities (e.g., endoxylanases, beta-xylosidases, arabinofuranosidases,alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, andalpha-glucuronyl esterases). Recent progress in assays of xylanolyticenzymes was summarized in several publications including Biely andPuchard, Recent progress in the assays of xylanolytic enzymes, 2006,Journal of the Science of Food and Agriculture 86(11): 1636-1647;Spanikova and Biely, 2006, Glucuronoyl esterase—Novel carbohydrateesterase produced by Schizophyllum commune, FEBS Letters 580(19):4597-4601; Herrmann, Vrsanska, Jurickova, Hirsch, Biely, and Kubicek,1997, The beta-D-xylosidase of Trichoderma reesei is a multifunctionalbeta-D-xylan xylohydrolase, Biochemical Journal 321: 375-381.

Total xylan degrading activity can be measured by determining thereducing sugars formed from various types of xylan, including, forexample, oat spelt, beechwood, and larchwood xylans, or by photometricdetermination of dyed xylan fragments released from various covalentlydyed xylans. The most common total xylanolytic activity assay is basedon production of reducing sugars from polymeric 4-O-methylglucuronoxylan as described in Bailey, Biely, Poutanen, 1992,Interlaboratory testing of methods for assay of xylanase activity,Journal of Biotechnology 23(3): 257-270. Xylanase activity can also bedetermined with 0.2% AZCL-arabinoxylan as substrate in 0.01% TRITON®X-100 (4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol) and 200mM sodium phosphate buffer pH 6 at 37° C. One unit of xylanase activityis defined as 1.0 μmole of azurine produced per minute at 37° C., pH 6from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6buffer.

For purposes of the present invention, xylan degrading activity isdetermined by measuring the increase in hydrolysis of birchwood xylan(Sigma Chemical Co., Inc., St. Louis, Mo., USA) by xylan-degradingenzyme(s) under the following typical conditions: 1 ml reactions, 5mg/ml substrate (total solids), 5 mg of xylanolytic protein/g ofsubstrate, 50 mM sodium acetate pH 5, 50° C., 24 hours, sugar analysisusing p-hydroxybenzoic acid hydrazide (PHBAH) assay as described byLever, 1972, A new reaction for colorimetric determination ofcarbohydrates, Anal. Biochem 47: 273-279.

Xylanase: The term “xylanase” means a 1,4-beta-D-xylan-xylohydrolase(E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1,4-beta-D-xylosidiclinkages in xylans. For purposes of the present invention, xylanaseactivity is determined with 0.2% AZCL-arabinoxylan as substrate in 0.01%TRITON® X-100 and 200 mM sodium phosphate buffer pH 6 at 37° C. One unitof xylanase activity is defined as 1.0 μmole of azurine produced perminute at 37° C., pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200mM sodium phosphate pH 6 buffer.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compositions comprising: (a) apolypeptide having cellulolytic enhancing activity; and (b) a liquor,wherein the combination of the polypeptide having cellulolytic enhancingactivity and the liquor enhances hydrolysis of a cellulosic material bya cellulolytic enzyme. In one aspect, the compositions further comprise(c) one or more (e.g., several) enzymes selected from the groupconsisting of a cellulase, a hemicellulase, an esterase, an expansin, alaccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease,and a swollenin.

The present invention also relates to methods for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellulolytic enhancing activity and a liquor, wherein thecombination of the polypeptide having cellulolytic enhancing activityand the liquor enhances hydrolysis of the cellulosic material by theenzyme composition. In one aspect, the method above further comprisesrecovering the degraded or converted cellulosic material. Solubleproducts of degradation or conversion of the cellulosic material can beseparated from the insoluble cellulosic material using technology wellknown in the art such as, for example, centrifugation, filtration, andgravity settling.

The present invention also relates to methods for producing afermentation product, comprising:

(a) saccharifying a cellulosic material with an enzyme composition inthe presence of a polypeptide having cellulolytic enhancing activity anda liquor, wherein the combination of the polypeptide having cellulolyticenhancing activity and the liquor enhances hydrolysis of the cellulosicmaterial by the enzyme composition;

(b) fermenting the saccharified cellulosic material with one or more(e.g., several) fermenting microorganisms to produce the fermentationproduct; and

(c) recovering the fermentation product from the fermentation.

The present invention also relates to methods of fermenting a cellulosicmaterial, comprising: fermenting the cellulosic material with one ormore (e.g., several) fermenting microorganisms, wherein the cellulosicmaterial is saccharified with an enzyme composition in the presence of apolypeptide having cellulolytic enhancing activity and a liquor, whereinthe combination of the polypeptide having cellulolytic enhancingactivity and the liquor enhances hydrolysis of the cellulosic materialby the enzyme composition. In one aspect, the fermenting of thecellulosic material produces a fermentation product. In another aspect,the method further comprises recovering the fermentation product fromthe fermentation.

Liquors

The term “liquor” means the solution phase, either aqueous, organic,ionic liquid, or combinations thereof, arising from treatment of alignocellulose and/or hemicellulose and/or lignacious material orfeedstock in a slurry, or monosaccharides thereof, e.g., xylose,arabinose, mannose, etc., under conditions as described herein, and thesoluble contents thereof. A liquor for cellulolytic enhancement of aGH61 polypeptide can be produced by treating a lignocellulosic,hemicellulosic, or lignacious material (or feedstock) by applying heatand/or pressure, optionally in the presence of a catalyst, e.g., acid,optionally in the presence of an organic solvent, and optionally incombination with physical disruption of the material, and thenseparating the solution from the residual solids. Alternatively, thelignocellulosic, hemicellulosic, or lignacious material can be slurriedand incubated in aqueous, organic, or ionic liquids or combinationsthereof as either solutions or suspensions without addition of heat orpressure. Such conditions determine the degree of cellulolyticenhancement obtainable through the combination of liquor and a GH61polypeptide during hydrolysis of a cellulosic substrate by a cellulasepreparation. The liquor can be separated from the treated material usingmethods standard in the art, such as filtration, sedimentation, orcentifugation.

In one aspect, the material for producing the liquor is herbaceousmaterial. In another aspect, the material is agricultural residue. Inanother aspect, the material is forestry residue. In another aspect, thematerial is municipal solid waste. In another aspect, the material iswaste paper. In another aspect, the material is pulp and paper millresidue. In another aspect, the material is pulping liquor. In anotheraspect, the material is mixed wood waste.

In another aspect, the material for producing the liquor is corn stover.In another aspect, the material is corn fiber. In another aspect, thematerial is corn cob. In another aspect, the material is orange peel. Inanother aspect, the material is rice straw. In another aspect, thematerial is wheat straw. In another aspect, the material is switchgrass. In another aspect, the material is miscanthus. In another aspect,the material is sugar cane bagasse. In another aspect, the material isenergy cane. In another aspect, the material is sorghum. In anotheraspect, the material is algae biomass.

In another aspect, the material for producing the liquor is softwood. Inanother aspect, the material is hardwood. In another aspect, thematerial is poplar. In another aspect, the material is pine. In anotheraspect, the material is spruce. In another aspect, the material is fir.In another aspect, the material is willow. In another aspect, thematerial is eucalyptus.

In another aspect, the material for producing the liquor is ahemicellulose. In another aspect, the material is a hemicellulose-richlignocellulose. In another aspect, the material is a xylan. In anotheraspect, the material is beechwood xylan. In another aspect, the materialis birch xylan. In another aspect, the material is spruce xylan. Inanother aspect, the material is arabinoxylan. In another aspect, thematerial is mannan. In another aspect, the material is glucomannan. Inanother aspect, the material contains beta-(1,4)-linked xylan. Inanother aspect, the material contains beta-(1,4)-linked mannan. Inanother aspect, the material contains branched beta-(1,4)-linked xylan,such as arabinoxylan and arabino-(glucoryono)-xylan. In another aspect,the material contains branched beta-(1,4)-linked mannan, such asgalactoglucomannan.

In another aspect, the material for producing the liquor is a C5monosaccharide (pentose). In another aspect, the material is arabinose.In another aspect, the material is xylose. In another aspect, thematerial is xylulose. In another aspect, the material is ribose. Inanother aspect, the material is ribulose. In another aspect, thematerial is acetyl-xylose. In another aspect, the material isferulyl-xylose. In another aspect, the material is glucurono-xylose ormethyl-glucurono-xylose.

In another aspect, the material for producing the liquor is a C6monosaccharide (hexose). In another aspect, the material is glucose. Inanother aspect, the material is mannose. In another aspect, the materialis fructose. In another aspect, the material is gulose. In anotheraspect, the material is allose. In another aspect, the material isaltrose. In another aspect, the material is idose. In another aspect,the material is talose. In another aspect, the material is galactose. Inanother aspect, the material is gluconic acid. In another aspect, thematerial is glucuronic acid. In another aspect, the material isgalactonic acid. In another aspect, the material is galacturonic acid.In another aspect, the material is psicose. In another aspect, thematerial is fructose. In another aspect, the material is sorbose. Inanother aspect, the material is tagatose.

In another aspect, the material for producing the liquor is a C4monosaccharide. In another aspect, the material is erythrose. In anotheraspect, the material is threose. In another aspect, the material iserythrulose.

In another aspect, the material for producing the liquor is a C3monosaccharide. In another aspect, the material is glyceraldehyde. Inanother aspect, the material is dihydroxyacetone.

In another aspect, the material for producing the liquor is lignin. Inanother aspect, the material is Kraft (Indulin) lignin. In anotheraspect, the material is p-hydroxyphenyl (H) rich lignin. In anotheraspect, the material is guaiacyl (G) rich lignin. In another aspect, thematerial is syringal (S) rich lignin. In another aspect, the material islignosulfonate. In another aspect the material is black liquor orcomponents thereof. In another aspect, the material is tall oil orcomponents thereof.

In another aspect, the material for producing the liquor is apost-pretreatment residue of biomass (the solid waste afterpretreatment. In another aspect, the material for producing the liquoris a post-saccharification residue of biomass (the solid waste aftersaccharification). In another aspect, the material for producing theliquor is a post-fermentation residue of biomass (the solid waste afterfermentation). In another aspect, the material for producing the liquoris a post-distillation residue of biomass (the solid waste afterdistillation).

In a non-limiting aspect, the material is treated using acid in therange of about 0.5 to about 5% (w/v), e.g., about 0.5 to about 4.5%(w/v), about 0.75 to about 4% (w/v), about 1.0 to about 3.5% (w/v),about 1.25 to about 3.0% (w/v), or about 1.5 to about 2.5% (w/v); a pHof about 0 to about 3, e.g., about 0.5 to about 3, about 0.5 to about2.5, about 1 to about 2, or about 1 to about 1.5; a time period of about1 to about 15 minutes, e.g., about 1 to about 12 minutes, about 2 toabout 10 minutes, about 3 to about 9 minutes, about 4 to about 9minutes, about 5 to about 9 minutes, or about 6 to about 8 minutes; at atemperature at about 130° C. to about 250° C., e.g., about 140° C. toabout 220° C., about 150° C. to about 200° C., about 160° C. to about190° C., about 160° C. to about 185° C., about 165° C. to about 180° C.,about 165° C. to about 175° C., or about 165° C. to about 170° C.; and apressure of about 100 to about 600 psi, e.g., about 50 to about 1700psi, about 100 to about 1500 psi, about 100 to about 1200 psi, about 100to about 1000 psi, about 100 to about 500 psi, about 100 to about 400psi, about 100 to about 300 psi, about 100 to about 200 psi, about 100to about 150 psi, or about 100 to about 120 psi. In another aspect, theacid is sulfuric acid. In another aspect, the acid is hydrochloric acid.In another aspect, the acid is nitric acid. In another aspect, the acidis phosphoric acid. In another aspect, the acid is acetic acid. Inanother aspect, the acid is citric acid. In another aspect, the acid issuccinic acid. In another aspect, the acid is tartaric acid. In anotheraspect, the acid is mixtures of any of the above acids. It is understoodherein that the conditions described above may need to be optimizeddepending on the material being treated and the reactor used to producethe liquor. Such optimization is well within the skill in the art.Conditions used to pretreat a cellulosic material as described hereinmay also be used to generate liquor from a particular feedstock.

In a preferred aspect, the material is treated using 1.4% (w/v) sulfuricacid for 8 minutes at 165° C. and 107 psi.

Treatment of a material to produce such liquor may also generate othercompounds that are inhibitory to cellulases and/or hemicellulases, e.g.,organic acids and lignin-derived compounds. Conditions can be selectedthat balance cellulolytic enhancing activity of a GH61 polypeptide withproduction of inhibitor compounds of cellulases and/or hemicellulases.Such conditions can vary depending on the material used for producingthe liquor. However, the liquor can be subjected to a molecular weightfilter(s) or dialysis, e.g. electrodialysis, using a membrane withnominal molecular weight cut-off of in the range of about 0.1 kDa toabout 10 kDa, e.g., about 0.5 kDa to about 7 kDa, about 0.5 kDa to about5 kDa, and about 1 kDa to about 3 kDa, to reduce the amount of theinhibitory compounds. Any method known in the art can be used to reducethe amount of the inhibitory compounds. In one aspect, the liquor isfurther processed to remove inhibitors of a cellulase, a hemicellulase,or a combination thereof.

In other aspects of the present invention, the liquor can be generatedin situ by pretreating a cellulosic material that will be saccharifiedby a cellulase preparation. However, in such instances the amount ofeffective liquor generated in situ may be insufficient with regard tothe GH61 polypeptide having cellulolytic enhancing activity,cellulolytic enzyme(s), and cellulose. For example, pretreatment of acellulosic material under mild conditions, e.g., auto-catalyzed steamexplosion, alkaline pretreatment, auto-hydrolysis, jet cooking, hotwater-pretreatment, organosolv using ethanol, glycerol, etc., diluteacid pretreatment, and the like (“low severity conditions”) or apretreatment that includes a wash or rinse step or unpretreatedcellulosic material compared to harsh conditions (“high severityconditions”) to produce in situ a liquor may be inadequate foroptimizing the cellulolytic enhancing activity of a GH61 polypeptide.Conditions employed to produce NREL preatreated corn stover, i.e., 1.4wt % sulfuric acid for 8 minutes at 165° C. and 107 psi (Example 1)would be considered high severity conditions. In such circumstances aliquor obtained using treatment conditions different from thepretreatment conditions of the cellulosic material can be added to thesaccharification reaction. In other aspects of the invention, alow-severity extraction treatment that extracts liquor can be usedinstead of conventional treatment techniques listed herein, and thisliquor can then be added to the saccharification reaction.

In one aspect, the liquor is obtained from a material that is the sameas the cellulosic material to be subjected to saccharification by acellulase composition. In another aspect, the liquor is obtained from amaterial that is different than the cellulosic material to be subjectedto saccharification by a cellulase composition.

In another aspect, the liquor is obtained from a material that is thesame as the cellulosic material to be subjected to saccharification by acellulase composition, but the treatment conditions used to produce theliquor are different from the pretreatment conditions of the cellulosicmaterial. In another aspect, the liquor is obtained from a material thatis the same as the cellulosic material to be subjected tosaccharification by a cellulase composition, and the treatmentconditions used to produce the liquor are the same as the pretreatmentconditions of the cellulosic material. In another aspect, the liquor isobtained from a material that is the same as the cellulosic material tobe subjected to saccharification by a cellulase composition, and thetreatment conditions used to produce the liquor are the same as thepretreatment conditions of the cellulosic material, and the liquor isfurther processed, e.g., concentrated, filtered to remove cellulaseinhibitors, filtered and concentrated, etc.

In another aspect, the liquor is obtained from a material that isdifferent than the cellulosic material to be subjected tosaccharification by a cellulase composition, and the treatmentconditions used to produce the liquor are different from thepretreatment conditions of the cellulosic material. In another aspect,the liquor is obtained from a material that is different than thecellulosic material to be subjected to saccharification by a cellulasecomposition, and the treatment conditions used to produce the liquor arethe same as the pretreatment conditions of the cellulosic material. Inanother aspect, the liquor is obtained from a material that is differentthan the cellulosic material to be subjected to saccharification by acellulase composition, and the treatment conditions used to produce theliquor are the same as the pretreatment conditions of the cellulosicmaterial, and the liquor is further processed, e.g., concentrated,filtered to remove cellulase inhibitors, filtered and concentrated, etc.Further processing to remove cellulose inhibitors can be accomplishedusing any method known in the art.

In another aspect, liquors generated in situ may be washed or dilutedand replaced with liquors generated ex situ to a greater or lesserextent, so the liquor composition/content is optimized for thecellulolytic enhancing effect of a GH61 polypeptide. In another aspect,the solids content of subsequent saccharifications is altered tooptimize the liquor content for the cellulolytic enhancing effect of aGH61 polypeptide.

The effective amount of the liquor can depend on one or more (e.g.,several) factors including, but not limited to, the mixture of componentcellulolytic enzymes, the cellulosic substrate, the concentration ofcellulosic substrate, the pretreatment(s) of the cellulosic substrate,non-cellulosic components (e.g., native or degraded lignin orhemicellulose), non-cellulase components, temperature, reaction time,and the liquor (e.g., filtered to remove cellulase and/or hemicellulaseinhibitors).

The liquor is preferably present in an amount that is not limiting withregard to the GH61 polypeptide having cellulolytic enhancing activity,cellulolytic enzyme(s), and cellulose. In one aspect, the liquor ispresent in an amount that is not limiting with regard to the GH61polypeptide having cellulolytic enhancing activity. In another aspect,the liquor is present in an amount that is not limiting with regard tothe cellulolytic enzyme(s). In another aspect, the liquor is present inan amount that is not limiting with regard to the cellulose. In anotheraspect, the liquor is present in an amount that is not limiting withregard to the GH61 polypeptide having cellulolytic enhancing activityand the cellulolytic enzyme(s). In another aspect, the liquor is presentin an amount that is not limiting with regard to the GH61 polypeptidehaving cellulolytic enhancing activity and the cellulose. In anotheraspect, the liquor is present in an amount that is not limiting withregard to the cellulolytic enzyme(s) and the cellulose. In anotheraspect, the liquor is present in an amount that is not limiting withregard to the GH61 polypeptide having cellulolytic enhancing activity,the cellulolytic enzyme(s), and the cellulose.

The liquor is preferably present in an amount that optimizes thecellulolytic enhancing activity of a GH61 polypeptide duringsaccharification with a cellulase composition. In one aspect, the liquoroptimizes the cellulolytic enhancing activity of a GH61 polypeptide witha GH61 effect as defined by Equation 3 (the ratio of fractionalhydrolysis in the presence to the absence of the GH61 polypeptide) ofpreferably at least 1.05, more preferably at least 1.10, more preferablyat least 1.15, more prefereably at least 1.2, more preferably at least1.25, more preferably at least 1.3, more preferably at least 1.35, morepreferably at least 1.4, more preferably at least 1.45, more preferablyat least 1.5, more preferably at least 1.55, more preferably at least1.6, more preferably at least 1.65, more preferably at least 1.7, morepreferably at least 1.75, more preferably at least 1.8, more preferablyat least 1.85, more preferably at least 1.9, most preferably at least1.95, and even most preferably at least 2. An increase in the GH61effect is obtained when liquor is added, relative to when liquor is notadded, during hydrolysis. In another aspect, the amount of GH61polypeptide is optimized for a given concentration of liquor. Suchoptimization is accomplished by varying the concentration of eachcomponent to determine the optimal ratio of the components duringsaccharification.

In another aspect, an effective amount of the liquor to cellulose isabout 10⁻⁶ to about 10 g per g of cellulose, e.g., about 10⁻⁶ to about7.5 g, about 10⁻⁶ to about 5, about 10⁻⁶ to about 2.5 g, about 10⁻⁶ toabout 1 g, about 10⁻⁵ to about 1 g, about 10⁻⁵ to about 10⁻¹ g, about10⁻⁴ to about 10⁻¹ g, about 10⁻³ to about 10⁻¹ g, and about 10⁻³ toabout 10⁻² g per g of cellulose.

In another aspect, the amount of liquor present that minimizesinhibition of a cellulase composition and in combination with a GH61polypeptide enhances hydrolysis by an enzyme composition is about 1 toabout 20% (v/v), e.g., about 1 to about 15%, about 1 to about 10%, about2 to about 7%, about 2 to about 5%, and about 3 to about 5%.

In the methods of the present invention, the term “liquor” encompassesone or more (e.g., several) liquors from different materials based onthe same or different conditions of treatment to produce the liquors.

In the methods of the present invention, the liquor is preferablypresent when a GH61 polypeptide is present, for example, is added withthe GH61 polypeptide. The liquor can also be added at different stagesof a saccharification. The liquor can also be redosed at differentstages of saccharification, e.g., daily, to maintain the presence of aneffective concentration of the liquor. The liquor can also be removed orwashed to various degrees, or may be diluted at different stages ofsaccharification. Liquors generated in situ may be washed and replacedwith liquors generated ex situ to a greater or lesser extent, at varioustimes during saccharification

In another aspect of the present invention, the liquor may be recycledfrom a completed saccharification or completed saccharification andfermentation to a new saccharification. The liquor can be recoveredusing standard methods in the art, e.g., filtration/centrifugation,sedimentation, and/or flocculation of solids materials pre- orpost-distillation, to remove residual solids, cellular debris, etc. andthen recirculated to the new saccharification.

Polypeptides Having Cellulolytic Enhancing Activity and PolynucleotidesThereof

In the methods of the present invention, any GH61 polypeptide havingcellulolytic enhancing activity can be used.

In a first aspect, the polypeptide having cellulolytic enhancingactivity comprises the following motifs:

(SEQ ID NO: 127 or SEQ ID NO: 128)[ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(4)-[HNQ] and[FW]-[TF]-K-[AIV],wherein X is any amino acid, X(4,5) is any amino acid at 4 or 5contiguous positions, and X(4) is any amino acid at 4 contiguouspositions.

The isolated polypeptide comprising the above-noted motifs may furthercomprise:

(SEQ ID NO: 129 or SEQ ID NO: 130) H-X(1,2)-G-P-X(3)-[YW]-[AILMV],(SEQ ID NO: 131) [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV], or(SEQ ID NO: 132 or SEQ ID NO: 133) H-X(1,2)-G-P-X(3)-[YW]-[AILMV] and(SEQ ID NO: 134) [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV],wherein X is any amino acid, X(1,2) is any amino acid at 1 position or 2contiguous positions, X(3) is any amino acid at 3 contiguous positions,and X(2) is any amino acid at 2 contiguous positions. In the abovemotifs, the accepted IUPAC single letter amino acid abbreviation isemployed.

In a preferred embodiment, the isolated GH61 polypeptide havingcellulolytic enhancing activity further comprisesH-X(1,2)-G-P-X(3)-[YW]-[AILMV] (SEQ ID NO: 135 or SEQ ID NO: 136). Inanother preferred embodiment, the isolated GH61 polypeptide havingcellulolytic enhancing activity further comprises[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO: 137). In anotherpreferred embodiment, the isolated GH61 polypeptide having cellulolyticenhancing activity further comprises H-X(1,2)-G-P-X(3)-[YW]-[AILMV] (SEQID NO: 138 or SEQ ID NO: 139) and[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO: 140).

In a second aspect, isolated polypeptides having cellulolytic enhancingactivity, comprise the following motif:

(SEQ ID NO: 141 or SEQ ID NO: 142)[ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(3)-A- [HNQ],wherein X is any amino acid, X(4,5) is any amino acid at 4 or 5contiguous positions, and X(3) is any amino acid at 3 contiguouspositions. In the above motif, the accepted IUPAC single letter aminoacid abbreviation is employed.

In a third aspect, the polypeptide having cellulolytic enhancingactivity comprises an amino acid sequence that has a degree of identityto the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ IDNO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44,SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO:54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ IDNO: 64, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150,SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ IDNO: 160, SEQ ID NO: 162, SEQ ID NO: 164, or SEQ ID NO: 166 of preferablyat least 60%, more preferably at least 65%, more preferably at least70%, more preferably at least 75%, more preferably at least 80%, morepreferably at least 85%, even more preferably at least 90%, mostpreferably at least 91%, at least 92%, at least 93%, at least 94%, or atleast 95%, or at least 100% and even most preferably at least 96%, atleast 97%, at least 98%, at least 99%, or at least 100%.

In a preferred aspect, the mature polypeptide is amino acids 20 to 326of SEQ ID NO: 2, amino acids 18 to 239 of SEQ ID NO: 4, amino acids 20to 258 of SEQ ID NO: 6, amino acids 19 to 226 of SEQ ID NO: 8, aminoacids 20 to 304 of SEQ ID NO: 10, amino acids 23 to 250 of SEQ ID NO:12, amino acids 22 to 249 of SEQ ID NO: 14, amino acids 20 to 249 of SEQID NO: 16, amino acids 18 to 232 of SEQ ID NO: 18, amino acids 16 to 235of SEQ ID NO: 20, amino acids 19 to 323 of SEQ ID NO: 22, amino acids 16to 310 of SEQ ID NO: 24, amino acids 20 to 246 of SEQ ID NO: 26, aminoacids 22 to 354 of SEQ ID NO: 28, amino acids 22 to 250 of SEQ ID NO:30, or amino acids 22 to 322 of SEQ ID NO: 32, amino acids 24 to 444 ofSEQ ID NO: 34, amino acids 26 to 253 of SEQ ID NO: 36, amino acids 20 to223 of SEQ ID NO: 38, amino acids 18 to 246 of SEQ ID NO: 40, aminoacids 20 to 334 of SEQ ID NO: 42, amino acids 18 to 227 of SEQ ID NO:44, amino acids 22 to 368 of SEQ ID NO: 46, amino acids 25 to 330 of SEQID NO: 48, amino acids 17 to 236 of SEQ ID NO: 50, amino acids 17 to 250of SEQ ID NO: 52, amino acids 23 to 478 of SEQ ID NO: 54, amino acids 17to 230 of SEQ ID NO: 56, amino acids 20 to 257 of SEQ ID NO: 58, aminoacids 23 to 251 of SEQ ID NO: 60, amino acids 19 to 349 of SEQ ID NO:62, amino acids 24 to 436 of SEQ ID NO: 64, amino acids 21 to 344 of SEQID NO: 144, amino acids 21 to 389 of SEQ ID NO: 146, amino acids 22 to406 of SEQ ID NO: 148, amino acids 20 to 427 of SEQ ID NO: 150, aminoacids 18 to 267 of SEQ ID NO: 152, amino acids 21 to 273 of SEQ ID NO:154, amino acids 21 to 322 of SEQ ID NO: 156, amino acids 18 to 234 ofSEQ ID NO: 158, amino acids 24 to 233 of SEQ ID NO: 160, amino acids 17to 237 of SEQ ID NO: 162, amino acids 20 to 484 of SEQ ID NO: 164, oramino acids 22 to 320 of SEQ ID NO: 166.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 2 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 2. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 2. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 326 of SEQ ID NO:2, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 326 of SEQ ID NO:2.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 4 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 4. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 4. In another preferred aspect, thepolypeptide comprises or consists of amino acids 18 to 239 of SEQ ID NO:4, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 18 to 239 of SEQ ID NO:4.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 6 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 6. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 6. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 258 of SEQ ID NO:6, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 258 of SEQ ID NO:6.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 8 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 8. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 8. In another preferred aspect, thepolypeptide comprises or consists of amino acids 19 to 226 of SEQ ID NO:8, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 19 to 226 of SEQ ID NO:8.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 10 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 10. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 10. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 304 of SEQ ID NO:10, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 304 of SEQ ID NO:10.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 12 or anallelic variant thereof; or a fragment thereof having cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 12. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 12. In another preferred aspect, thepolypeptide comprises or consists of amino acids 16 to 317 of SEQ ID NO:12, or an allelic variant thereof; or a fragment thereof havingcellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 16 to 317 of SEQ ID NO:12.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 14 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 14. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 14. In another preferred aspect, thepolypeptide comprises or consists of amino acids 23 to 250 of SEQ ID NO:14, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 23 to 250 of SEQ ID NO:14.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 16 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 16. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 16. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 249 of SEQ ID NO:16, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 249 of SEQ ID NO:16.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 18 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 18. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 18. In another preferred aspect, thepolypeptide comprises or consists of amino acids 18 to 232 of SEQ ID NO:18, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 18 to 232 of SEQ ID NO:18.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 20 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 20. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 20. In another preferred aspect, thepolypeptide comprises or consists of amino acids 16 to 235 of SEQ ID NO:20, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 16 to 235 of SEQ ID NO:20.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 22 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 22. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 22. In another preferred aspect, thepolypeptide comprises or consists of amino acids 19 to 323 of SEQ ID NO:22, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 19 to 323 of SEQ ID NO:22.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 24 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 24. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 24. In another preferred aspect, thepolypeptide comprises or consists of amino acids 16 to 310 of SEQ ID NO:24, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 16 to 310 of SEQ ID NO:24.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 26 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 26. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 26. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 246 of SEQ ID NO:26, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 246 of SEQ ID NO:26.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 28 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 28. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 28. In another preferred aspect, thepolypeptide comprises or consists of amino acids 22 to 354 of SEQ ID NO:28, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 22 to 354 of SEQ ID NO:28.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 30 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 30. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 30. In another preferred aspect, thepolypeptide comprises or consists of amino acids 22 to 250 of SEQ ID NO:30, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 22 to 250 of SEQ ID NO:30.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 32 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 32. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 32. In another preferred aspect, thepolypeptide comprises or consists of amino acids 22 to 322 of SEQ ID NO:32, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 22 to 322 of SEQ ID NO:32.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 34 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 34. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 34. In another preferred aspect, thepolypeptide comprises or consists of amino acids 24 to 444 of SEQ ID NO:34, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 24 to 444 of SEQ ID NO:34.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 36 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 36. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 36. In another preferred aspect, thepolypeptide comprises or consists of amino acids 26 to 253 of SEQ ID NO:36, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 26 to 253 of SEQ ID NO:36.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 38 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 38. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 38. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 223 of SEQ ID NO:38, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 223 of SEQ ID NO:38.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 40 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 40. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 40. In another preferred aspect, thepolypeptide comprises or consists of amino acids 18 to 246 of SEQ ID NO:40, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 18 to 246 of SEQ ID NO:40.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 42 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 42. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 42. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 334 of SEQ ID NO:42, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 334 of SEQ ID NO:42.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 44 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 44. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 44. In another preferred aspect, thepolypeptide comprises or consists of amino acids 18 to 227 of SEQ ID NO:44, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 18 to 227 of SEQ ID NO:44.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 46 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 46. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 46. In another preferred aspect, thepolypeptide comprises or consists of amino acids 22 to 368 of SEQ ID NO:46, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 22 to 368 of SEQ ID NO:46.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 48 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 48. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 48. In another preferred aspect, thepolypeptide comprises or consists of amino acids 25 to 330 of SEQ ID NO:48, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 25 to 330 of SEQ ID NO:48.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 50 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 50. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 50. In another preferred aspect, thepolypeptide comprises or consists of amino acids 17 to 236 of SEQ ID NO:50, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 17 to 236 of SEQ ID NO:50.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 52 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 52. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 52. In another preferred aspect, thepolypeptide comprises or consists of amino acids 19 to 250 of SEQ ID NO:52, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 19 to 250 of SEQ ID NO:52.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 54 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 54. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 54. In another preferred aspect, thepolypeptide comprises or consists of amino acids 23 to 478 of SEQ ID NO:54, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 23 to 478 of SEQ ID NO:54.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 56 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 56. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 56. In another preferred aspect, thepolypeptide comprises or consists of amino acids 17 to 230 of SEQ ID NO:56, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 17 to 230 of SEQ ID NO:56.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 58 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 58. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 58. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 257 of SEQ ID NO:58, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 257 of SEQ ID NO:58.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 60 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 60. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 60. In another preferred aspect, thepolypeptide comprises or consists of amino acids 23 to 251 of SEQ ID NO:60, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 23 to 251 of SEQ ID NO:60.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 62 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 62. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 62. In another preferred aspect, thepolypeptide comprises or consists of amino acids 19 to 349 of SEQ ID NO:62, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 19 to 349 of SEQ ID NO:62.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 64 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 64. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 64. In another preferred aspect, thepolypeptide comprises or consists of amino acids 24 to 436 of SEQ ID NO:64, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 24 to 436 of SEQ ID NO:64.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 144 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 144. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 144. In another preferred aspect, thepolypeptide comprises or consists of amino acids 21 to 344 of SEQ ID NO:144, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 21 to 344 of SEQ ID NO:144.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 146 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 146. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 146. In another preferred aspect, thepolypeptide comprises or consists of amino acids 21 to 389 of SEQ ID NO:146, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 21 to 389 of SEQ ID NO:146.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 148 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 148. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 148. In another preferred aspect, thepolypeptide comprises or consists of amino acids 22 to 406 of SEQ ID NO:148, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 22 to 406 of SEQ ID NO:148.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 150 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 150. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 150. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 427 of SEQ ID NO:150, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 427 of SEQ ID NO:150.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 152 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 152. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 152. In another preferred aspect, thepolypeptide comprises or consists of amino acids 18 to 267 of SEQ ID NO:152, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 18 to 267 of SEQ ID NO:152.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 154 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 154. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 154. In another preferred aspect, thepolypeptide comprises or consists of amino acids 21 to 273 of SEQ ID NO:154, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 21 to 273 of SEQ ID NO:154.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 156 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 156. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 156. In another preferred aspect, thepolypeptide comprises or consists of amino acids 21 to 322 of SEQ ID NO:156, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 21 to 322 of SEQ ID NO:156.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 158 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 158. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 158. In another preferred aspect, thepolypeptide comprises or consists of amino acids 18 to 234 of SEQ ID NO:158, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 18 to 234 of SEQ ID NO:158.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 160 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 160. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 160. In another preferred aspect, thepolypeptide comprises or consists of amino acids 24 to 233 of SEQ ID NO:160, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 24 to 233 of SEQ ID NO:160.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 162 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 162. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 162. In another preferred aspect, thepolypeptide comprises or consists of amino acids 17 to 237 of SEQ ID NO:162, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 17 to 237 of SEQ ID NO:162.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 164 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 164. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 164. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 484 of SEQ ID NO:164, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 20 to 484 of SEQ ID NO:164.

A polypeptide having cellulolytic enhancing activity preferablycomprises or consists of the amino acid sequence of SEQ ID NO: 166 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In a preferred aspect, the polypeptide comprises orconsists of the amino acid sequence of SEQ ID NO: 166. In anotherpreferred aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 166. In another preferred aspect, thepolypeptide comprises or consists of amino acids 22 to 320 of SEQ ID NO:166, or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide comprises or consists of amino acids 22 to 320 of SEQ ID NO:166.

Preferably, a fragment of the mature polypeptide of SEQ ID NO: 2contains at least 277 amino acid residues, more preferably at least 287amino acid residues, and most preferably at least 297 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:4 contains at least 185 amino acid residues, more preferably at least195 amino acid residues, and most preferably at least 205 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:6 contains at least 200 amino acid residues, more preferably at least212 amino acid residues, and most preferably at least 224 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:8 contains at least 175 amino acid residues, more preferably at least185 amino acid residues, and most preferably at least 195 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:10 contains at least 240 amino acid residues, more preferably at least255 amino acid residues, and most preferably at least 270 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:12 contains at least 255 amino acid residues, more preferably at least270 amino acid residues, and most preferably at least 285 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:14 contains at least 175 amino acid residues, more preferably at least190 amino acid residues, and most preferably at least 205 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:16 contains at least 200 amino acid residues, more preferably at least210 amino acid residues, and most preferably at least 220 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:18 contains at least 185 amino acid residues, more preferably at least195 amino acid residues, and most preferably at least 205 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:20 contains at least 190 amino acid residues, more preferably at least200 amino acid residues, and most preferably at least 210 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:22 contains at least 260 amino acid residues, more preferably at least275 amino acid residues, and most preferably at least 290 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:24 contains at least 250 amino acid residues, more preferably at least265 amino acid residues, and most preferably at least 280 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:26 contains at least 195 amino acid residues, more preferably at least205 amino acid residues, and most preferably at least 214 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:28 contains at least 285 amino acid residues, more preferably at least300 amino acid residues, and most preferably at least 315 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:30 contains at least 200 amino acid residues, more preferably at least210 amino acid residues, and most preferably at least 220 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:32 contains at least 255 amino acid residues, more preferably at least270 amino acid residues, and most preferably at least 285 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:34 contains at least 360 amino acid residues, more preferably at least380 amino acid residues, and most preferably at least 400 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:36 contains at least 200 amino acid residues, more preferably at least210 amino acid residues, and most preferably at least 220 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:38 contains at least 170 amino acid residues, more preferably at least180 amino acid residues, and most preferably at least 190 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:40 contains at least 190 amino acid residues, more preferably at least200 amino acid residues, and most preferably at least 210 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:42 contains at least 265 amino acid residues, more preferably at least280 amino acid residues, and most preferably at least 295 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:44 contains at least 180 amino acid residues, more preferably at least190 amino acid residues, and most preferably at least 200 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:46 contains at least 320 amino acid residues, more preferably at least335 amino acid residues, and most preferably at least 350 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:48 contains at least 255 amino acid residues, more preferably at least270 amino acid residues, and most preferably at least 285 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:50 contains at least 190 amino acid residues, more preferably at least200 amino acid residues, and most preferably at least 210 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:52 contains at least 200 amino acid residues, more preferably at least210 amino acid residues, and most preferably at least 220 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:54 contains at least 380 amino acid residues, more preferably at least400 amino acid residues, and most preferably at least 420 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:56 contains at least 180 amino acid residues, more preferably at least190 amino acid residues, and most preferably at least 200 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:58 contains at least 210 amino acid residues, more preferably at least220 amino acid residues, and most preferably at least 230 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:60 contains at least 190 amino acid residues, more preferably at least200 amino acid residues, and most preferably at least 210 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:62 contains at least 270 amino acid residues, more preferably at least290 amino acid residues, and most preferably at least 310 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:64 contains at least 340 amino acid residues, more preferably at least360 amino acid residues, and most preferably at least 380 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:144 contains at least 280 amino acid residues, more preferably at least295 amino acid residues, and most preferably at least 310 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:146 contains at least 310 amino acid residues, more preferably at least330 amino acid residues, and most preferably at least 350 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:148 contains at least 320 amino acid residues, more preferably at least340 amino acid residues, and most preferably at least 360 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:150 contains at least 350 amino acid residues, more preferably at least370 amino acid residues, and most preferably at least 390 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:152 contains at least 220 amino acid residues, more preferably at least230 amino acid residues, and most preferably at least 240 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:154 contains at least 220 amino acid residues, more preferably at least230 amino acid residues, and most preferably at least 240 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:156 contains at least 255 amino acid residues, more preferably at least270 amino acid residues, and most preferably at least 285 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:158 contains at least 185 amino acid residues, more preferably at least195 amino acid residues, and most preferably at least 205 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:160 contains at least 180 amino acid residues, more preferably at least190 amino acid residues, and most preferably at least 200 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:162 contains at least 190 amino acid residues, more preferably at least200 amino acid residues, and most preferably at least 210 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:164 contains at least 385 amino acid residues, more preferably at least410 amino acid residues, and most preferably at least 435 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:166 contains at least 255 amino acid residues, more preferably at least270 amino acid residues, and most preferably at least 285 amino acidresidues.

Preferably, a subsequence of the mature polypeptide coding sequence ofSEQ ID NO: 1 contains at least 831 nucleotides, more preferably at least861 nucleotides, and most preferably at least 891 nucleotides.Preferably, a subsequence of the mature polypeptide coding sequence ofSEQ ID NO: 3 contains at least 555 nucleotides, more preferably at least585 nucleotides, and most preferably at least 615 nucleotides.Preferably, a subsequence of the mature polypeptide coding sequence ofSEQ ID NO: 5 contains at least 600 nucleotides, more preferably at least636 nucleotides, and most preferably at least 672 nucleotides.Preferably, a subsequence of the mature polypeptide coding sequence ofSEQ ID NO: 7 contains at least 525 nucleotides, more preferably at least555 nucleotides, and most preferably at least 585 nucleotides.Preferably, a subsequence of the mature polypeptide coding sequence ofSEQ ID NO: 9 contains at least 720 nucleotides, more preferably at least765 nucleotides, and most preferably at least 810 nucleotides.Preferably, a subsequence of the mature polypeptide coding sequence ofSEQ ID NO: 11 contains at least 765 nucleotides, more preferably atleast 810 nucleotides, and most preferably at least 855 nucleotides.Preferably, a subsequence of the mature polypeptide coding sequence ofnucleotides 67 to 796 of SEQ ID NO: 13 contains at least 525nucleotides, more preferably at least 570 nucleotides, and mostpreferably at least 615 nucleotides. Preferably, a subsequence of themature polypeptide coding sequence of SEQ ID NO: 15 contains at least600 nucleotides, more preferably at least 630 nucleotides, and mostpreferably at least 660 nucleotides. Preferably, a subsequence of themature polypeptide coding sequence of SEQ ID NO: 17 contains at least555 nucleotides, more preferably at least 585 nucleotides, and mostpreferably at least 615 nucleotides. Preferably, a subsequence of themature polypeptide coding sequence of SEQ ID NO: 19 contains at least570 nucleotides, more preferably at least 600 nucleotides, and mostpreferably at least 630 nucleotides. Preferably, a subsequence of themature polypeptide coding sequence of SEQ ID NO: 21 contains at least780 nucleotides, more preferably at least 825 nucleotides, and mostpreferably at least 870 nucleotides. Preferably, a subsequence of themature polypeptide coding sequence of SEQ ID NO: 23 contains at least750 nucleotides, more preferably at least 795 nucleotides, and mostpreferably at least 840 nucleotides. Preferably, a subsequence of themature polypeptide coding sequence of SEQ ID NO: 25 contains at least585 nucleotides, more preferably at least 615 nucleotides, and mostpreferably at least 645 nucleotides. Preferably, a subsequence of themature polypeptide coding sequence of SEQ ID NO: 27 contains at least855 nucleotides, more preferably at least 900 nucleotides, and mostpreferably at least 945 nucleotides. Preferably, a subsequence of themature polypeptide coding sequence of SEQ ID NO: 29 contains at least600 nucleotides, more preferably at least 630 nucleotides, and mostpreferably at least 660 nucleotides. Preferably, a subsequence of themature polypeptide coding sequence of SEQ ID NO: 31 contains at least765 nucleotides, more preferably at least 810 nucleotides, and mostpreferably at least 855 nucleotides. Preferably, a subsequence of themature polypeptide coding sequence of SEQ ID NO: 33 contains at least1180 nucleotides, more preferably at least 1140 nucleotides, and mostpreferably at least 1200 nucleotides. Preferably, a subsequence of themature polypeptide coding sequence of SEQ ID NO: 35 contains at least600 nucleotides, more preferably at least 630 nucleotides, and mostpreferably at least 660 nucleotides. Preferably, a subsequence of themature polypeptide coding sequence of SEQ ID NO: 37 contains at least170 amino acid residues, more preferably at least 180 amino acidresidues, and most preferably at least 190 nucleotides. Preferably, asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 39contains at least 570 nucleotides, more preferably at least 600nucleotides, and most preferably at least 630 nucleotides. Preferably, asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 41contains at least 795 nucleotides, more preferably at least 840nucleotides, and most preferably at least 885 nucleotides. Preferably, asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 43contains at least 540 nucleotides, more preferably at least 570nucleotides, and most preferably at least 600 nucleotides. Preferably, asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 45contains at least 960 nucleotides, more preferably at least 1005nucleotides, and most preferably at least 1050 nucleotides. Preferably,a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 47contains at least 765 nucleotides, more preferably at least 810nucleotides, and most preferably at least 855 nucleotides. Preferably, asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 49contains at least 570 nucleotides, more preferably at least 600nucleotides, and most preferably at least 630 nucleotides. Preferably, asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 51contains at least 600 nucleotides, more preferably at least 630nucleotides, and most preferably at least 660 nucleotides. Preferably, asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 53contains at least 1140 nucleotides, more preferably at least 1200nucleotides, and most preferably at least 1260 nucleotides. Preferably,a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 55contains at least 540 nucleotides, more preferably at least 570nucleotides, and most preferably at least 600 nucleotides. Preferably, asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 57contains at least 630 nucleotides, more preferably at least 690nucleotides, and most preferably at least 720 nucleotides. Preferably, asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 59contains at least 570 nucleotides, more preferably at least 600nucleotides, and most preferably at least 630 nucleotides. Preferably, asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 61contains at least 810 nucleotides, more preferably at least 870nucleotides, and most preferably at least 930 nucleotides. Preferably, asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 63contains at least 1020 nucleotides, more preferably at least 1080nucleotides, and most preferably at least 1140 nucleotides. Preferably,a subsequence of the mature polypeptide coding sequence of SEQ ID NO:143 contains at least 840 nucleotides, more preferably at least 885nucleotides, and most preferably at least 930 nucleotides. Preferably, asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 145contains at least 930 nucleotides, more preferably at least 960nucleotides, and most preferably at least 1050 nucleotides. Preferably,a subsequence of the mature polypeptide coding sequence of SEQ ID NO:147 contains at least 960 nucleotides, more preferably at least 1020nucleotides, and most preferably at least 1080 nucleotides. Preferably,a subsequence of the mature polypeptide coding sequence of SEQ ID NO:149 contains at least 1050 nucleotides, more preferably at least 1110nucleotides, and most preferably at least 1170 nucleotides. Preferably,a subsequence of the mature polypeptide coding sequence of SEQ ID NO:151 contains at least 660 nucleotides, more preferably at least 690nucleotides, and most preferably at least 720 nucleotides. Preferably, asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 153contains at least 660 nucleotides, more preferably at least 690nucleotides, and most preferably at least 720 nucleotides. Preferably, asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 155contains at least 765 nucleotides, more preferably at least 810nucleotides, and most preferably at least 855 nucleotides. Preferably, asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 157contains at least 555 nucleotides, more preferably at least 585nucleotides, and most preferably at least 615 nucleotides. Preferably, asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 159contains at least 540 nucleotides, more preferably at least 570nucleotides, and most preferably at least 600 nucleotides. Preferably, asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 161contains at least 570 nucleotides, more preferably at least 600nucleotides, and most preferably at least 630 nucleotides. Preferably, asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 163contains at least 1155 nucleotides, more preferably at least 1230nucleotides, and most preferably at least 1305 nucleotides. Preferably,a subsequence of the mature polypeptide coding sequence of SEQ ID NO:165 contains at least 765 nucleotides, more preferably at least 810nucleotides, and most preferably at least 855 nucleotides.

In a fourth aspect, the polypeptide having cellulolytic enhancingactivity is encoded by a polynucleotide that hybridizes under at leastvery low stringency conditions, preferably at least low stringencyconditions, more preferably at least medium stringency conditions, morepreferably at least medium-high stringency conditions, even morepreferably at least high stringency conditions, and most preferably atleast very high stringency conditions with (i) the mature polypeptidecoding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ IDNO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45,SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO:55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ IDNO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151,SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ IDNO: 161, or SEQ ID NO: 163, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:11, SEQ ID NO: 15, SEQ ID NO: 155, SEQ ID NO: 157, or SEQ ID NO: 159, orthe cDNA sequence of the mature polypeptide coding sequence of SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 17, SEQ IDNO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37,SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO:47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ IDNO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 143, SEQID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO:153, SEQ ID NO: 161, SEQ ID NO: 163, or SEQ ID NO: 165, (iii) asubsequence of (i) or (ii), or (iv) a full-length complementary strandof (i), (ii), or (iii) (J. Sambrook, E. F. Fritsch, and T. Maniatus,1989, supra). A subsequence of the mature polypeptide coding sequence ofSEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO:19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ IDNO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47,SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO:57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 143, SEQ IDNO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153,SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, or SEQID NO: 163 contains at least 100 contiguous nucleotides or preferably atleast 200 contiguous nucleotides. Moreover, the subsequence may encode apolypeptide fragment that has cellulolytic enhancing activity. In apreferred aspect, the mature polypeptide coding sequence is nucleotides388 to 1332 of SEQ ID NO: 1, nucleotides 98 to 821 of SEQ ID NO: 3,nucleotides 126 to 978 of SEQ ID NO: 5, nucleotides 55 to 678 of SEQ IDNO: 7, nucleotides 58 to 912 of SEQ ID NO: 9, nucleotides 46 to 951 ofSEQ ID NO: 11, nucleotides 67 to 796 of SEQ ID NO: 13, nucleotides 77 to766 of SEQ ID NO: 15, nucleotides 52 to 921 of SEQ ID NO: 17,nucleotides 46 to 851 of SEQ ID NO: 19, nucleotides 55 to 1239 of SEQ IDNO: 21, nucleotides 46 to 1250 of SEQ ID NO: 23, nucleotides 58 to 811of SEQ ID NO: 25, nucleotides 64 to 1112 of SEQ ID NO: 27, nucleotides64 to 859 of SEQ ID NO: 29, nucleotides 64 to 1018 of SEQ ID NO: 31,nucleotides 70 to 1483 of SEQ ID NO: 33, nucleotides 76 to 832 of SEQ IDNO: 35, nucleotides 58 to 974 of SEQ ID NO: 37, nucleotides 52 to 875 ofSEQ ID NO: 39, nucleotides 58 to 1250 of SEQ ID NO: 41, nucleotides 52to 795 of SEQ ID NO: 43, nucleotides 64 to 1104 of SEQ ID NO: 45,nucleotides 73 to 990 of SEQ ID NO: 47, nucleotides 49 to 1218 of SEQ IDNO: 49, nucleotides 55 to 930 of SEQ ID NO: 51, nucleotides 67 to 1581of SEQ ID NO: 53, nucleotides 49 to 865 of SEQ ID NO: 55, nucleotides 58to 1065 of SEQ ID NO: 57, nucleotides 67 to 868 of SEQ ID NO: 59,nucleotides 55 to 1099 of SEQ ID NO: 61, nucleotides 70 to 1483 of SEQID NO: 63, nucleotides 61 to 1032 of SEQ ID NO: 143, nucleotides 61 to1167 of SEQ ID NO: 145, nucleotides 64 to 1218 of SEQ ID NO: 147,nucleotides 58 to 1281 of SEQ ID NO: 149, nucleotides 52 to 801 of SEQID NO: 151, nucleotides 61 to 819 of SEQ ID NO: 153, nucleotides 61 to966 of SEQ ID NO: 155, nucleotides 52 to 702 of SEQ ID NO: 157,nucleotides 70 to 699 of SEQ ID NO: 159, nucleotides 49 to 711 of SEQ IDNO: 161, nucleotides 76 to 1452 of SEQ ID NO: 163, or nucleotides 64 to1018 of SEQ ID NO: 165.

The nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25,SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO:35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ IDNO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63,SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ IDNO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159,SEQ ID NO: 161, or SEQ ID NO: 163, or a subsequence thereof; as well asthe amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16,SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO:26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ IDNO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54,SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO:64, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO:160, SEQ ID NO: 162, SEQ ID NO: 164, or SEQ ID NO: 166, or a fragmentthereof, may be used to design a nucleic acid probe to identify andclone DNA encoding polypeptides having cellulolytic enhancing activityfrom strains of different genera or species according to methods wellknown in the art. In particular, such probes can be used forhybridization with the genomic DNA or cDNA of the genus or species ofinterest, following standard Southern blotting procedures, in order toidentify and isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least14, preferably at least 25, more preferably at least 35, and mostpreferably at least 70 nucleotides in length. It is, however, preferredthat the nucleic acid probe is at least 100 nucleotides in length. Forexample, the nucleic acid probe may be at least 200 nucleotides,preferably at least 300 nucleotides, more preferably at least 400nucleotides, or most preferably at least 500 nucleotides in length. Evenlonger probes may be used, e.g., nucleic acid probes that are preferablyat least 600 nucleotides, more preferably at least 700 nucleotides, evenmore preferably at least 800 nucleotides, or most preferably at least900 nucleotides in length. Both DNA and RNA probes can be used. Theprobes are typically labeled for detecting the corresponding gene (forexample, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes areencompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may,therefore, be screened for DNA that hybridizes with the probes describedabove and encodes a polypeptide having cellulolytic enhancing activity.Genomic or other DNA from such other strains may be separated by agaroseor polyacrylamide gel electrophoresis, or other separation techniques.DNA from the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA that is homologous with SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ IDNO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49,SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO:59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 143, SEQ ID NO: 145, SEQ IDNO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155,SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, or SEQ ID NO: 163, or asubsequence thereof, the carrier material is preferably used in aSouthern blot.

For purposes of the present invention, hybridization indicates that thenucleotide sequence hybridizes to a labeled nucleic acid probecorresponding to the mature polypeptide coding sequence of SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ IDNO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49,SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO:59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 143, SEQ ID NO: 145, SEQ IDNO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155,SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, or SEQ ID NO: 163; thegenomic DNA sequence of the mature polypeptide coding sequence of SEQ IDNO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 155, SEQID NO: 157, or SEQ ID NO: 159, or the cDNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO:23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ IDNO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51,SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO:61, SEQ ID NO: 63, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 161, SEQ ID NO:163, or SEQ ID NO: 165; the full-length complementary strand thereof; ora subsequence thereof, under very low to very high stringencyconditions, as described supra.

In a preferred aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 1. In another preferred aspect, thenucleic acid probe is nucleotides 388 to 1332 of SEQ ID NO: 1. Inanother preferred aspect, the nucleic acid probe is a polynucleotidesequence that encodes the polypeptide of SEQ ID NO: 2, or a subsequencethereof. In another preferred aspect, the nucleic acid probe is SEQ IDNO: 1. In another preferred aspect, the nucleic acid probe is thepolynucleotide sequence contained in plasmid pEJG120 which is containedin E. coli NRRL B-30699, wherein the polynucleotide sequence thereofencodes a polypeptide having cellulolytic enhancing activity. In anotherpreferred aspect, the nucleic acid probe is the mature polypeptidecoding sequence contained in plasmid pEJG120 which is contained in E.coli NRRL B-30699.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 3. In another preferredaspect, the nucleic acid probe is nucleotides 98 to 821 of SEQ ID NO: 3.In another preferred aspect, the nucleic acid probe is a polynucleotidesequence that encodes the polypeptide of SEQ ID NO: 4, or a subsequencethereof. In another preferred aspect, the nucleic acid probe is SEQ IDNO: 3. In another preferred aspect, the nucleic acid probe is thepolynucleotide sequence contained in plasmid pTter61C which is containedin E. coli NRRL B-30813, wherein the polynucleotide sequence thereofencodes a polypeptide having cellulolytic enhancing activity. In anotherpreferred aspect, the nucleic acid probe is the mature polypeptidecoding sequence contained in plasmid pTter61C which is contained in E.coli NRRL B-30813.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 5. In another preferredaspect, the nucleic acid probe is nucleotides 126 to 978 of SEQ ID NO:5. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 6, ora subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 5. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pTter61D whichis contained in E. coli NRRL B-30812, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pTter61D whichis contained in E. coli NRRL B-30812.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 7. In another preferredaspect, the nucleic acid probe is nucleotides 55 to 678 of SEQ ID NO: 7.In another preferred aspect, the nucleic acid probe is a polynucleotidesequence that encodes the polypeptide of SEQ ID NO: 8, or a subsequencethereof. In another preferred aspect, the nucleic acid probe is SEQ IDNO: 7. In another preferred aspect, the nucleic acid probe is thepolynucleotide sequence contained in plasmid pTter61E which is containedin E. coli NRRL B-30814, wherein the polynucleotide sequence thereofencodes a polypeptide having cellulolytic enhancing activity. In anotherpreferred aspect, the nucleic acid probe is the mature polypeptidecoding sequence contained in plasmid pTter61E which is contained in E.coli NRRL B-30814.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 9. In another preferredaspect, the nucleic acid probe is nucleotides 58 to 912 of SEQ ID NO: 9In another preferred aspect, the nucleic acid probe is a polynucleotidesequence that encodes the polypeptide of SEQ ID NO: 10, or a subsequencethereof. In another preferred aspect, the nucleic acid probe is SEQ IDNO: 9. In another preferred aspect, the nucleic acid probe is thepolynucleotide sequence contained in plasmid pTter61G which is containedin E. coli NRRL B-30811, wherein the polynucleotide sequence thereofencodes a polypeptide having cellulolytic enhancing activity. In anotherpreferred aspect, the nucleic acid probe is the mature polypeptidecoding sequence contained in plasmid pTter61G which is contained in E.coli NRRL B-30811.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 11. In another preferredaspect, the nucleic acid probe is nucleotides 46 to 951 of SEQ ID NO:11. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 12,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 11. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pTter61F whichis contained in E. coli NRRL B-50044, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding region contained in plasmid pTter61F which iscontained in E. coli NRRL B-50044.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 13. In another preferredaspect, the nucleic acid probe is nucleotides 67 to 796 of SEQ ID NO:13. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 14,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 13. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pDZA2-7 whichis contained in E. coli NRRL B-30704, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pDZA2-7 which iscontained in E. coli NRRL B-30704.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 15. In another preferredaspect, the nucleic acid probe is nucleotides 77 to 766 of SEQ ID NO:15. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 16,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 15. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pTr3337 whichis contained in E. coli NRRL B-30878, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pTr3337 which iscontained in E. coli NRRL B-30878.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 17. In another preferredaspect, the nucleic acid probe is nucleotides 52 to 921 of SEQ ID NO:17. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 18,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 17. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pSMai190 whichis contained in E. coli NRRL B-50084, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pSMai190 whichis contained in E. coli NRRL B-50084.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 19. In another preferredaspect, the nucleic acid probe is nucleotides 46 to 851 of SEQ ID NO:19. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 20,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 19. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pSMai192 whichis contained in E. coli NRRL B-50086, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pSMai192 whichis contained in E. coli NRRL B-50086.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 21. In another preferredaspect, the nucleic acid probe is nucleotides 55 to 1239 of SEQ ID NO:21. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 22,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 21. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pSMai191 whichis contained in E. coli NRRL B-50085, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pSMai191 whichis contained in E. coli NRRL B-50085.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 23. In another preferredaspect, the nucleic acid probe is nucleotides 46 to 1250 of SEQ ID NO:23. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 24,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 23. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pSMai193 whichis contained in E. coli NRRL B-50087, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pSMai193 whichis contained in E. coli NRRL B-50087.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 25. In another preferredaspect, the nucleic acid probe is nucleotides 58 to 811 of SEQ ID NO:25. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 26,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 25. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pSMai187 whichis contained in E. coli NRRL B-50083, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pSMai187 whichis contained in E. coli NRRL B-50083.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 27. In another preferredaspect, the nucleic acid probe is nucleotides 64 to 1112 of SEQ ID NO:27. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 28,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 27. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pXYZ1473 whichis contained in E. coli DSM 22075, wherein the polynucleotide sequencethereof encodes a polypeptide having cellulolytic enhancing activity. Inanother preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence contained in plasmid pXYZ1473 which iscontained in E. coli DSM 22075.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 29. In another preferredaspect, the nucleic acid probe is nucleotides 64 to 859 of SEQ ID NO:29. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 30,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 29.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 31. In another preferredaspect, the nucleic acid probe is nucleotides 64 to 1018 of SEQ ID NO:31. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 32,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 31. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pGEM-T-Ppin7which is contained in E. coli DSM 22711, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pGEM-T-Ppin7which is contained in E. coli DSM 22711.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 33. In another preferredaspect, the nucleic acid probe is nucleotides 70 to 1483 of SEQ ID NO:33. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 34,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 33. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pXYZ1483 whichis contained in E. coli DSM 22600, wherein the polynucleotide sequencethereof encodes a polypeptide having cellulolytic enhancing activity. Inanother preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence contained in plasmid pXYZ1483 which iscontained in E. coli DSM 22600.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 35. In another preferredaspect, the nucleic acid probe is nucleotides 76 to 832 of SEQ ID NO:35. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 36,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 35. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmidpGEM-T-GH61D23Y4 which is contained in E. coli DSM 22882, wherein thepolynucleotide sequence thereof encodes a polypeptide havingcellulolytic enhancing activity. In another preferred aspect, thenucleic acid probe is the mature polypeptide coding sequence containedin plasmid pGEM-T-GH61D23Y4 which is contained in E. coli DSM 22882.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 37. In another preferredaspect, the nucleic acid probe is nucleotides 58 to 974 of SEQ ID NO:37. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 38,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 37. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pSMai213 whichis contained in E. coli NRRL B-50300, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pSMai213 whichis contained in E. coli NRRL B-50300.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 39. In another preferredaspect, the nucleic acid probe is nucleotides 52 to 875 of SEQ ID NO:39. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 40,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 39. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pSMai216 whichis contained in E. coli NRRL B-50301, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pSMai216 whichis contained in E. coli NRRL B-50301.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 41. In another preferredaspect, the nucleic acid probe is nucleotides 58 to 1250 of SEQ ID NO:41. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 42,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 41. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid p pSMai217which is contained in E. coli NRRL B-50302, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pSMai217 whichis contained in E. coli NRRL B-50302.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 43. In another preferredaspect, the nucleic acid probe is nucleotides 52 to 795 of SEQ ID NO:43. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 44,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 43. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pSMai218 whichis contained in E. coli NRRL B-50303, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pSMai218 whichis contained in E. coli NRRL B-50303.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 45. In another preferredaspect, the nucleic acid probe is nucleotides 64 to 1104 of SEQ ID NO:45. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 46,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 45. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pAG68 which iscontained in E. coli NRRL B-50320, wherein the polynucleotide sequencethereof encodes a polypeptide having cellulolytic enhancing activity. Inanother preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence contained in plasmid pAG68 which iscontained in E. coli NRRL B-50320.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 47. In another preferredaspect, the nucleic acid probe is nucleotides 73 to 990 of SEQ ID NO:47. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 48,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 47. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pAG69 which iscontained in E. coli NRRL B-50321, wherein the polynucleotide sequencethereof encodes a polypeptide having cellulolytic enhancing activity. Inanother preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence contained in plasmid pAG69 which iscontained in E. coli NRRL B-50321.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 49. In another preferredaspect, the nucleic acid probe is nucleotides 49 to 1218 of SEQ ID NO:49. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 50,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 49. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pAG75 which iscontained in E. coli NRRL B-50322, wherein the polynucleotide sequencethereof encodes a polypeptide having cellulolytic enhancing activity. Inanother preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence contained in plasmid pAG75 which iscontained in E. coli NRRL B-50322.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 51. In another preferredaspect, the nucleic acid probe is nucleotides 55 to 930 of SEQ ID NO:51. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 52,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 51. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pAG76 which iscontained in E. coli NRRL B-50323, wherein the polynucleotide sequencethereof encodes a polypeptide having cellulolytic enhancing activity. Inanother preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence contained in plasmid pAG76 which iscontained in E. coli NRRL B-50323.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 53. In another preferredaspect, the nucleic acid probe is nucleotides 67 to 1581 of SEQ ID NO:53. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 54,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 53. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pAG77 which iscontained in E. coli NRRL B-50324, wherein the polynucleotide sequencethereof encodes a polypeptide having cellulolytic enhancing activity. Inanother preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence contained in plasmid pAG77 which iscontained in E. coli NRRL B-50324.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 55. In another preferredaspect, the nucleic acid probe is nucleotides 49 to 865 of SEQ ID NO:55. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 56,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 55. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pAG78 which iscontained in E. coli NRRL B-50325, wherein the polynucleotide sequencethereof encodes a polypeptide having cellulolytic enhancing activity. Inanother preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence contained in plasmid pAG78 which iscontained in E. coli NRRL B-50325.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 57. In another preferredaspect, the nucleic acid probe is nucleotides 58 to 1065 of SEQ ID NO:57. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 58,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 57. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid p pAG79 whichis contained in E. coli NRRL B-50326, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pAG79 which iscontained in E. coli NRRL B-50326.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 59. In another preferredaspect, the nucleic acid probe is nucleotides 67 to 868 of SEQ ID NO:59. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 60,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 59. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid plasmidpGEM-T-GH61a51486 which is contained in E. coli DSM 22656, wherein thepolynucleotide sequence thereof encodes a polypeptide havingcellulolytic enhancing activity. In another preferred aspect, thenucleic acid probe is the mature polypeptide coding sequence containedin plasmid plasmid pGEM-T-GH61a51486 which is contained in E. coli DSM22656.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 61. In another preferredaspect, the nucleic acid probe is nucleotides 55 to 1099 of SEQ ID NO:61. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 62,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 61. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pGEM-T-GH61DYFwhich is contained in E. coli DSM 22654, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pGEM-T-GH61DYFwhich is contained in E. coli DSM 22654.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 63. In another preferredaspect, the nucleic acid probe is nucleotides 70 to 1483 of SEQ ID NO:63. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 64,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 63. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmidpGEM-T-GH61D14YH which is contained in E. coli DSM 22657, wherein thepolynucleotide sequence thereof encodes a polypeptide havingcellulolytic enhancing activity. In another preferred aspect, thenucleic acid probe is the mature polypeptide coding sequence containedin plasmid pGEM-T-GH61D14YH which is contained in E. coli DSM 22657.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 143. In another preferredaspect, the nucleic acid probe is nucleotides 61 to 1032 of SEQ ID NO:143. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 143,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 143. In another preferred aspect, the nucleic acidprobe is the mature polypeptide coding sequence of SEQ ID NO: 145. Inanother preferred aspect, the nucleic acid probe is nucleotides 61 to1167 of SEQ ID NO: 145. In another preferred aspect, the nucleic acidprobe is a polynucleotide sequence that encodes the polypeptide of SEQID NO: 145, or a subsequence thereof. In another preferred aspect, thenucleic acid probe is SEQ ID NO: 145.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 147. In another preferredaspect, the nucleic acid probe is nucleotides 64 to 1218 of SEQ ID NO:147. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 147,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 147. In another preferred aspect, the nucleic acidprobe is the mature polypeptide coding sequence of SEQ ID NO: 149. Inanother preferred aspect, the nucleic acid probe is nucleotides 58 to1281 of SEQ ID NO: 149. In another preferred aspect, the nucleic acidprobe is a polynucleotide sequence that encodes the polypeptide of SEQID NO: 149, or a subsequence thereof. In another preferred aspect, thenucleic acid probe is SEQ ID NO: 149.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 151. In another preferredaspect, the nucleic acid probe is nucleotides 52 to 801 of SEQ ID NO:151. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 151,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 151.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 153. In another preferredaspect, the nucleic acid probe is nucleotides 61 to 819 of SEQ ID NO:153. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 153,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 153.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 155. In another preferredaspect, the nucleic acid probe is nucleotides 61 to 966 of SEQ ID NO:155. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 155,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 155.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 157. In another preferredaspect, the nucleic acid probe is nucleotides 52 to 702 of SEQ ID NO:157. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 157,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 157.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 159. In another preferredaspect, the nucleic acid probe is nucleotides 70 to 699 of SEQ ID NO:159. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 159,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 159.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 161. In another preferredaspect, the nucleic acid probe is nucleotides 49 to 711 of SEQ ID NO:161. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 161,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 161.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 163. In another preferredaspect, the nucleic acid probe is nucleotides 76 to 1452 of SEQ ID NO:163. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 163,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 163.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 165. In another preferredaspect, the nucleic acid probe is nucleotides 64 to 1018 of SEQ ID NO:165. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 165,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 165.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5× SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and either 25% formamide for very lowand low stringencies, 35% formamide for medium and medium-highstringencies, or 50% formamide for high and very high stringencies,following standard Southern blotting procedures for 12 to 24 hoursoptimally. The carrier material is finally washed three times each for15 minutes using 2× SSC, 0.2% SDS at 45° C. (very low stringency), at50° C. (low stringency), at 55° C. (medium stringency), at 60° C.(medium-high stringency), at 65° C. (high stringency), and at 70° C.(very high stringency).

For short probes of about 15 nucleotides to about 70 nucleotides inlength, stringency conditions are defined as prehybridization andhybridization at about 5° C. to about 10° C. below the calculated T_(m)using the calculation according to Bolton and McCarthy (1962, Proc.Natl. Acad. Sci. USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6mM EDTA, 0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA perml following standard Southern blotting procedures for 12 to 24 hoursoptimally. The carrier material is finally washed once in 6× SCC plus0.1% SDS for 15 minutes and twice each for 15 minutes using 6× SSC at 5°C. to 10° C. below the calculated T_(m).

In a fifth aspect, the polypeptide having cellulolytic enhancingactivity is encoded by a polynucleotide comprising or consisting of anucleotide sequence that has a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15,SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO:25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ IDNO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53,SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO:63, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO:159, SEQ ID NO: 161, or SEQ ID NO: 163 of preferably at least 60%, morepreferably at least 65%, more preferably at least 70%, more preferablyat least 75%, more preferably at least 80%, more preferably at least85%, even more preferably at least 90%, most preferably at least 91%, atleast 92%, at least 93%, at least 94%, or at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, at least 99%, or atleast 100%.

In a sixth aspect, the polypeptide having cellulolytic enhancingactivity is an artificial variant comprising a substitution, deletion,and/or insertion of one or more (e.g., several) amino acids of themature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14, SEQ ID NO: 16,SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO:26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ IDNO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54,SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO:64, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO:160, SEQ ID NO: 162, SEQ ID NO: 164, or SEQ ID NO: 166; or a homologoussequence thereof. Preferably, amino acid changes are of a minor nature,that is conservative amino acid substitutions or insertions that do notsignificantly affect the folding and/or activity of the protein; smalldeletions, typically of one to about 30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to about 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a parent polypeptide can be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for cellulolytic enhancingactivity to identify amino acid residues that are critical to theactivity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem.271: 4699-4708. The active site of the enzyme or other biologicalinteraction can also be determined by physical analysis of structure, asdetermined by such techniques as nuclear magnetic resonance,crystallography, electron diffraction, or photoaffinity labeling, inconjunction with mutation of putative contact site amino acids. See, forexample, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992,J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identities of essential amino acids can also be inferred fromanalysis of identities with polypeptides that are related to the parentpolypeptide.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14,SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO:24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ IDNO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52,SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO:62, SEQ ID NO: 64, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO:158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, or SEQ ID NO: 166,is not more than 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9.

A polypeptide having cellulolytic enhancing activity may be obtainedfrom microorganisms of any genus. For purposes of the present invention,the term “obtained from” as used herein in connection with a givensource shall mean that the polypeptide encoded by a polynucleotide isproduced by the source or by a strain in which the polynucleotide fromthe source has been inserted. In one aspect, the polypeptide obtainedfrom a given source is secreted extracellularly.

A polypeptide having cellulolytic enhancing activity may be a bacterialpolypeptide. For example, the polypeptide may be a gram positivebacterial polypeptide such as a Bacillus, Streptococcus, Streptomyces,Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium,Geobacillus, or Oceanobacillus polypeptide having cellulolytic enhancingactivity, or a Gram negative bacterial polypeptide such as an E. coli,Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium,Fusobacterium, Ilyobacter, Neisseria, or Ureaplasma polypeptide havingcellulolytic enhancing activity.

In one aspect, the polypeptide is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having cellulolytic enhancing activity.

In another aspect, the polypeptide is a Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equisubsp. Zooepidemicus polypeptide having cellulolytic enhancing activity.

In another aspect, the polypeptide is a Streptomyces achromogenes,Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus,or Streptomyces lividans polypeptide having cellulolytic enhancingactivity.

The polypeptide having cellulolytic enhancing activity may also be afungal polypeptide, and more preferably a yeast polypeptide such as aCandida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia polypeptide having cellulolytic enhancing activity; or morepreferably a filamentous fungal polypeptide such as aan Acremonium,Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria,Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus,Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus,Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides,Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus,Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora,Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia,Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum,Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylariapolypeptide having cellulolytic enhancing activity.

In another aspect, the polypeptide is a Saccharomyces carlsbergensis,Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomycesdouglasii, Saccharomyces kluyveri, Saccharomyces norbensis, orSaccharomyces oviformis polypeptide having cellulolytic enhancingactivity.

In another aspect, the polypeptide is an Acremonium cellulolyticus,Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus,Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Chrysosporium keratinophilum,Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporiummerdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporiumqueenslandicum, Chrysosporium zonatum, Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsuiphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,lrpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium funiculosum, Penicillium pinophilum, Penicilliumpurpurogenum, Phanerochaete chrysosporium, Thielavia achromatica,Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis,Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielaviaperuviana, Thielavia spededonium, Thielavia setosa, Thielaviasubthermophila, Thielavia terrestris, Trichoderma harzianum, Trichodermakoningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichodermaviride, or Trichophaea saccata polypeptide having cellulolytic enhancingactivity.

It will be understood that for the aforementioned species the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, polypeptides having cellulolytic enhancing activity may beidentified and obtained from other sources including microorganismsisolated from nature (e.g., soil, composts, water, etc.) using theabove-mentioned probes. Techniques for isolating microorganisms fromnatural habitats are well known in the art. The polynucleotide may thenbe obtained by similarly screening a genomic DNA or cDNA library of sucha microorganism. Once a polynucleotide encoding a polypeptide has beendetected with the probe(s), the polynucleotide can be isolated or clonedby utilizing techniques that are well known to those of ordinary skillin the art (see, e.g., Sambrook et al., 1989, supra)

Polynucleotides comprising nucleotide sequences that encode polypeptidehaving cellulolytic enhancing activity can be isolated and utilized toexpress the polypeptide having cellulolytic enhancing activity forevaluation in the methods of the present invention.

The techniques used to isolate or clone a polynucleotide encoding apolypeptide are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of thepolynucleotides from such genomic DNA can be effected, e.g., by usingthe well known polymerase chain reaction (PCR) or antibody screening ofexpression libraries to detect cloned DNA fragments with sharedstructural features. See, e.g., Innis et al., 1990, PCR: A Guide toMethods and Application, Academic Press, New York. Other nucleic acidamplification procedures such as ligase chain reaction (LCR), ligationactivated transcription (LAT) and polynucleotide-based amplification(NASBA) may be used. The polynucleotides may be cloned from a strain ora related organism and thus, for example, may be an allelic or speciesvariant of the polypeptide encoding region of the polynucleotide.

The polynucleotides comprise nucleotide sequences that have a degree ofidentity to the mature polypeptide coding sequence of SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21,SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO:31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ IDNO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59,SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO:147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, or SEQ ID NO: 163 ofpreferably at least 60%, more preferably at least 65%, more preferablyat least 70%, more preferably at least 75%, more preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 91%, at least 92%, at least 93%, at least 94%,or at least 95%, and even most preferably at least 96%, at least 97%, atleast 98%, or at least 99%, which encode a polypeptide havingcellulolytic enhancing activity.

The polynucleotide may also be a polynucleotide encoding a polypeptidehaving cellulolytic enhancing activity that hybridizes under at leastvery low stringency conditions, preferably at least low stringencyconditions, more preferably at least medium stringency conditions, morepreferably at least medium-high stringency conditions, even morepreferably at least high stringency conditions, and most preferably atleast very high stringency conditions with (i) the mature polypeptidecoding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ IDNO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45,SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO:55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ IDNO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151,SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ IDNO: 161, or SEQ ID NO: 163, (ii) the genomic DNA sequence of the maturepolypeptide coding sequence of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:11, SEQ ID NO: 15, SEQ ID NO: 155, SEQ ID NO: 157, or SEQ ID NO: 159 orthe cDNA sequence of the mature polypeptide coding sequence of SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 17, SEQ IDNO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37,SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO:47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ IDNO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 143, SEQID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO:153, SEQ ID NO: 161, SEQ ID NO: 163, or SEQ ID NO: 165, or (iii) afull-length complementary strand of (i) or (ii); or allelic variants andsubsequences thereof (Sambrook et al., 1989, supra), as defined herein.

As described earlier, the techniques used to isolate or clone apolynucleotide encoding a polypeptide are known in the art and includeisolation from genomic DNA, preparation from cDNA, or a combinationthereof.

Enzyme Compositions

The enzyme compositions can comprise any protein that is useful indegrading or converting a cellulosic material.

In one aspect, the enzyme composition comprises or further comprises oneor more (e.g., several) proteins selected from the group consisting of acellulase, a hemicellulase, an esterase, an expansin, a laccase, aligninolytic enzyme, a pectinase, a peroxidase, a protease, and aswollenin. In another aspect, the cellulase is preferably one or more(e.g., several) enzymes selected from the group consisting of anendoglucanase, a cellobiohydrolase, and a beta-glucosidase. In anotheraspect, the hemicellulase is preferably one or more (e.g., several)enzymes selected from the group consisting of an acetylmannan esterase,an acetylxylan esterase, an arabinanase, an arabinofuranosidase, acoumaric acid esterase, a feruloyl esterase, a galactosidase, aglucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase, axylanase, and a xylosidase.

In another aspect, the enzyme composition comprises one or more (e.g.,several) cellulolytic enzymes. In another aspect, the enzyme compositioncomprises or further comprises one or more (e.g., several)hemicellulolytic enzymes. In another aspect, the enzyme compositioncomprises one or more (e.g., several) cellulolytic enzymes and one ormore (e.g., several) hemicellulolytic enzymes. In another aspect, theenzyme composition comprises one or more (e.g., several) enzymesselected from the group of cellulolytic enzymes and hemicellulolyticenzymes. In another aspect, the enzyme composition comprises anendoglucanase. In another aspect, the enzyme composition comprises acellobiohydrolase. In another aspect, the enzyme composition comprises abeta-glucosidase. In another aspect, the enzyme composition comprises anendoglucanase and a cellobiohydrolase. In another aspect, the enzymecomposition comprises an endoglucanase and a beta-glucosidase. Inanother aspect, the enzyme composition comprises a cellobiohydrolase anda beta-glucosidase. In another aspect, the enzyme composition comprisesan endoglucanase, a cellobiohydrolase, and a beta-glucosidase.

In another aspect, the enzyme composition comprises an acetylmannanesterase. In another aspect, the enzyme composition comprises anacetylxylan esterase. In another aspect, the enzyme compositioncomprises an arabinanase (e.g., alpha-L-arabinanase). In another aspect,the enzyme composition comprises an arabinofuranosidase (e.g.,alpha-L-arabinofuranosidase). In another aspect, the enzyme compositioncomprises a coumaric acid esterase. In another aspect, the enzymecomposition comprises a feruloyl esterase. In another aspect, the enzymecomposition comprises a galactosidase (e.g., alpha-galactosidase and/orbeta-galactosidase). In another aspect, the enzyme composition comprisesa glucuronidase (e.g., alpha-D-glucuronidase). In another aspect, theenzyme composition comprises a glucuronoyl esterase. In another aspect,the enzyme composition comprises a mannanase. In another aspect, theenzyme composition comprises a mannosidase (e.g., beta-mannosidase). Inanother aspect, the enzyme composition comprises a xylanase. In apreferred aspect, the xylanase is a Family 10 xylanase. In anotheraspect, the enzyme composition comprises a xylosidase (e.g.,beta-xylosidase).

In another aspect, the enzyme composition comprises an esterase. Inanother aspect, the enzyme composition comprises an expansin. In anotheraspect, the enzyme composition comprises a laccase. In another aspect,the enzyme composition comprises a ligninolytic enzyme. In a preferredaspect, the ligninolytic enzyme is a manganese peroxidase. In anotherpreferred aspect, the ligninolytic enzyme is a lignin peroxidase. Inanother preferred aspect, the ligninolytic enzyme is a H₂O₂-producingenzyme. In another aspect, the enzyme composition comprises a pectinase.In another aspect, the enzyme composition comprises a peroxidase. Inanother aspect, the enzyme composition comprises a protease. In anotheraspect, the enzyme composition comprises a swollenin.

In the methods of the present invention, the enzyme(s) can be addedprior to or during fermentation, e.g., during saccharification or duringor after propagation of the fermenting microorganism(s).

One or more (e.g., several) components of the enzyme composition may bewild-type proteins, recombinant proteins, or a combination of wild-typeproteins and recombinant proteins. For example, one or more (e.g.,several) components may be native proteins of a cell, which is used as ahost cell to express recombinantly one or more (e.g., several) othercomponents of the enzyme composition. One or more (e.g., several)components of the enzyme composition may be produced as monocomponents,which are then combined to form the enzyme composition. The enzymecomposition may be a combination of multicomponent and monocomponentprotein preparations.

The enzymes used in the methods of the present invention may be in anyform suitable for use, such as, for example, a crude fermentation brothwith or without cells removed, a cell lysate with or without cellulardebris, a semi-purified or purified enzyme preparation, or a host cellas a source of the enzymes. The enzyme composition may be a dry powderor granulate, a non-dusting granulate, a liquid, a stabilized liquid, ora stabilized protected enzyme. Liquid enzyme preparations may, forinstance, be stabilized by adding stabilizers such as a sugar, a sugaralcohol or another polyol, and/or lactic acid or another organic acidaccording to established processes.

The enzymes can be derived or obtained from any suitable origin,including, bacterial, fungal, yeast, plant, or mammalian origin. Theterm “obtained” means herein that the enzyme may have been isolated froman organism that naturally produces the enzyme as a native enzyme. Theterm “obtained” also means herein that the enzyme may have been producedrecombinantly in a host organism employing methods described herein,wherein the recombinantly produced enzyme is either native or foreign tothe host organism or has a modified amino acid sequence, e.g., havingone or more (e.g., several) amino acids that are deleted, insertedand/or substituted, i.e., a recombinantly produced enzyme that is amutant and/or a fragment of a native amino acid sequence or an enzymeproduced by nucleic acid shuffling processes known in the art.Encompassed within the meaning of a native enzyme are natural variantsand within the meaning of a foreign enzyme are variants obtainedrecombinantly, such as by site-directed mutagenesis or shuffling.

The polypeptide having enzyme activity may be a bacterial polypeptide.For example, the polypeptide may be a gram positive bacterialpolypeptide such as a Bacillus, Streptococcus, Streptomyces,Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium,Geobacillus, or Oceanobacillus polypeptide having enzyme activity, or aGram negative bacterial polypeptide such as an E. coli, Pseudomonas,Salmonella, Campylobacter, Helicobacter, Flavobacterium, Fusobacterium,Ilyobacter, Neisseria, or Ureaplasma polypeptide having enzyme activity.

In a preferred aspect, the polypeptide is a Bacillus alkalophilus,Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having enzyme activity.

In another preferred aspect, the polypeptide is a Streptococcusequisimilis, Streptococcus pyogenes, Streptococcus uberis, orStreptococcus equi subsp. Zooepidemicus polypeptide having enzymeactivity.

In another preferred aspect, the polypeptide is a Streptomycesachromogenes, Streptomyces avermitilis, Streptomyces coelicolor,Streptomyces griseus, or Streptomyces lividans polypeptide having enzymeactivity.

The polypeptide having enzyme activity may also be a fungal polypeptide,and more preferably a yeast polypeptide such as a Candida,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiapolypeptide having enzyme activity; or more preferably a filamentousfungal polypeptide such as an Acremonium, Agaricus, Alternaria,Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium,Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes,Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium,Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula,Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvariella, or Xylaria polypeptide having enzymeactivity.

In a preferred aspect, the polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis polypeptide having enzymeactivity.

In another preferred aspect, the polypeptide is an Acremoniumcellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillusfumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillusnidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporiumkeratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum,Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium zonatum, Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicolainsolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum,Penicillium purpurogenum, Phanerochaete chrysosporium, Thielaviaachromatica, Thielavia albomyces, Thielavia albopilosa, Thielaviaaustraleinsis, Thielavia fimeti, Thielavia microspora, Thielaviaovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa,Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,Trichoderma viride, or Trichophaea saccata polypeptide having enzymeactivity.

Chemically modified or protein engineered mutants of the polypeptideshaving enzyme activity may also be used.

One or more (e.g., several) components of the enzyme composition may bea recombinant component, i.e., produced by cloning of a DNA sequenceencoding the single component and subsequent cell transformed with theDNA sequence and expressed in a host (see, for example, WO 91/17243 andWO 91/17244). The host is preferably a heterologous host (enzyme isforeign to host), but the host may under certain conditions also be ahomologous host (enzyme is native to host). Monocomponent cellulolyticenzymes may also be prepared by purifying such a protein from afermentation broth.

In one aspect, the one or more (e.g., several) cellulolytic enzymescomprise a commercial cellulolytic enzyme preparation. Examples ofcommercial cellulolytic enzyme preparations suitable for use in thepresent invention include, for example, CELLIC™ CTec (Novozymes A/S),CELLIC™ CTec2 (Novozymes A/S), CELLUCLAST™ (Novozymes A/S), NOVOZYM™ 188(Novozymes A/S), CELLUZYME™ (Novozymes A/S), CEREFLO™ (Novozymes A/S),and ULTRAFLO™ (Novozymes A/S), ACCELERASE™ (Genencor Int.), LAMINEX™(Genencor Int.), SPEZYME™ CP (Genencor Int.), FILTRASE® NL (DSM);METHAPLUS® S/L 100 (DSM), ROHAMENT™ 7069 W (Röhm GmbH), FIBREZYME® LDI(Dyadic International, Inc.), FIBREZYME® LBR (Dyadic International,Inc.), or VISCOSTAR® 150L (Dyadic International, Inc.). The cellulaseenzymes are added in amounts effective from about 0.001 to about 5.0 wt% of solids, more preferably from about 0.025 to about 4.0 wt % ofsolids, and most preferably from about 0.005 to about 2.0 wt % ofsolids. The cellulase enzymes are added in amounts effective from about0.001 to about 5.0 wt % of solids, more preferably from about 0.025 toabout 4.0 wt % of solids, and most preferably from about 0.005 to about2.0 wt % of solids.

Examples of bacterial endoglucanases that can be used in the methods ofthe present invention, include, but are not limited to, an Acidothermuscellulolyticus endoglucanase (WO 91/05039; WO 93/15186; U.S. Pat. No.5,275,944; WO 96/02551; U.S. Pat. No. 5,536,655, WO 00/70031, WO05/093050); Thermobifida fusca endoglucanase III (WO 05/093050); andThermobifida fusca endoglucanase V (WO 05/093050).

Examples of fungal endoglucanases that can be used in the presentinvention include, but are not limited to, a Trichoderma reeseiendoglucanase I (Penttila et al., 1986, Gene 45: 253-263; Trichodermareesei Cel7B endoglucanase I; GENBANK™ accession no. M15665; SEQ ID NO:66); Trichoderma reesei endoglucanase II (Saloheimo, et al., 1988, Gene63:11-22; Trichoderma reesei Cel5A endoglucanase II; GENBANK™ accessionno. M19373; SEQ ID NO: 68); Trichoderma reesei endoglucanase III (Okadaet al., 1988, Appl. Environ. Microbiol. 64: 555-563; GENBANK™ accessionno. AB003694; SEQ ID NO: 70); Trichoderma reesei endoglucanase V(Saloheimo et al., 1994, Molecular Microbiology 13: 219-228; GENBANK™accession no. Z33381; SEQ ID NO: 72); Aspergillus aculeatusendoglucanase (Ooi et al., 1990, Nucleic Acids Research 18: 5884);Aspergillus kawachii endoglucanase (Sakamoto et al., 1995, CurrentGenetics 27: 435-439); Erwinia carotovara endoglucanase (Saarilahti etal., 1990, Gene 90: 9-14); Fusarium oxysporum endoglucanase (GENBANK™accession no. L29381); Humicola grisea var. thermoidea endoglucanase(GENBANK™ accession no. AB003107); Melanocarpus albomyces endoglucanase(GENBANK™ accession no. MAL515703); Neurospora crassa endoglucanase(GENBANK™ accession no. XM_324477); Humicola insolens endoglucanase V(SEQ ID NO: 74); Myceliophthora thermophila CBS 117.65 endoglucanase(SEQ ID NO: 76); basidiomycete CBS 495.95 endoglucanase (SEQ ID NO: 78);basidiomycete CBS 494.95 endoglucanase (SEQ ID NO: 80); Thielaviaterrestris NRRL 8126 CEL6B endoglucanase (SEQ ID NO: 82); Thielaviaterrestris NRRL 8126 CEL6C endoglucanase (SEQ ID NO: 84); Thielaviaterrestris NRRL 8126 CEL7C endoglucanase (SEQ ID NO: 86); Thielaviaterrestris NRRL 8126 CEL7E endoglucanase (SEQ ID NO: 88); Thielaviaterrestris NRRL 8126 CEL7F endoglucanase (SEQ ID NO: 90); Cladorrhinumfoecundissimum ATCC 62373 CEL7A endoglucanase (SEQ ID NO: 92); andTrichoderma reesei strain No. VTT-D-80133 endoglucanase (SEQ ID NO: 94;GENBANK™ accession no. M15665). The endoglucanases of SEQ ID NO: 66, SEQID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76,SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO:86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, and SEQ ID NO: 94described above are encoded by the mature polypeptide coding sequence ofSEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO:73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ IDNO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, andSEQ ID NO: 93, respectively.

Examples of cellobiohydrolases useful in the present invention include,but are not limited to, Trichoderma reesei cellobiohydrolase I (SEQ IDNO: 96); Trichoderma reesei cellobiohydrolase II (SEQ ID NO: 98);Humicola insolens cellobiohydrolase I (SEQ ID NO: 100); Myceliophthorathermophila cellobiohydrolase II (SEQ ID NO: 102 and SEQ ID NO: 104);Thielavia terrestris cellobiohydrolase II (CEL6A) (SEQ ID NO: 106);Chaetomium thermophilum cellobiohydrolase I (SEQ ID NO: 108); andChaetomium thermophilum cellobiohydrolase II (SEQ ID NO: 110). Thecellobiohydrolases of SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO:110, and SEQ ID NO: 112 described above are encoded by the maturepolypeptide coding sequence of SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO:101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, and SEQ ID NO: 109,respectively.

Examples of beta-glucosidases useful in the present invention include,but are not limited to, Aspergillus oryzae beta-glucosidase (SEQ ID NO:112); Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 114);Penicillium brasilianum IBT 20888 beta-glucosidase (SEQ ID NO: 116);Aspergillus niger beta-glucosidase (SEQ ID NO: 118); and Aspergillusaculeatus beta-glucosidase (SEQ ID NO: 120). The beta-glucosidases ofSEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, and SEQID NO: 120 described above are encoded by the mature polypeptide codingsequence of SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO:117, and SEQ ID NO: 119, respectively.

Examples of other beta-glucosidases useful in the present inventioninclude a Aspergillus oryzae beta-glucosidase variant fusion protein ofSEQ ID NO: 122 or the Aspergillus oryzae beta-glucosidase fusion proteinof SEQ ID NO: 124. The beta-glucosidase fusion proteins of SEQ ID NO:122 and SEQ ID NO: 124 are encoded by SEQ ID NO: 121 and SEQ ID NO: 123,respectively.

The Aspergillus oryzae polypeptide having beta-glucosidase activity canbe obtained according to WO 2002/095014. The Aspergillus fumigatuspolypeptide having beta-glucosidase activity can be obtained accordingto WO 2005/047499. The Penicillium brasilianum polypeptide havingbeta-glucosidase activity can be obtained according to WO 2007/019442.The Aspergillus niger polypeptide having beta-glucosidase activity canbe obtained according to Dan et al., 2000, J. Biol. Chem. 275:4973-4980. The Aspergillus aculeatus polypeptide having beta-glucosidaseactivity can be obtained according to Kawaguchi et al., 1996, Gene 173:287-288.

Other useful endoglucanases, cellobiohydrolases, and beta-glucosidasesare disclosed in numerous Glycosyl Hydrolase families using theclassification according to Henrissat B., 1991, A classification ofglycosyl hydrolases based on amino-acid sequence similarities, Biochem.J. 280: 309-316, and Henrissat B., and Bairoch A., 1996, Updating thesequence-based classification of glycosyl hydrolases, Biochem. J. 316:695-696.

Other cellulolytic enzymes that may be useful in the present inventionare described in EP 495,257, EP 531,315, EP 531,372, WO 89/09259, WO94/07998, WO 95/24471, WO 96/11262, WO 96/29397, WO 96/034108, WO97/14804, WO 98/08940, WO 98/012307, WO 98/13465, WO 98/015619, WO98/015633, WO 98/028411, WO 99/06574, WO 99/10481, WO 99/025846, WO99/025847, WO 99/031255, WO 2000/009707, WO 2002/050245, WO2002/0076792, WO 2002/101078, WO 2003/027306, WO 2003/052054, WO2003/052055, WO 2003/052056, WO 2003/052057, WO 2003/052118, WO2004/016760, WO 2004/043980, WO 2004/048592, WO 2005/001065, WO2005/028636, WO 2005/093050, WO 2005/093073, WO 2006/074005, WO2006/117432, WO 2007/071818, WO 2007/071820, WO 2008/008070, WO2008/008793, U.S. Pat. Nos. 4,435,307, 5,457,046, 5,648,263, 5,686,593,5,691,178, 5,763,254, and 5,776,757.

In one aspect, the one or more (e.g., several) hemicellulolytic enzymescomprise a commercial hemicellulolytic enzyme preparation. Examples ofcommercial hemicellulolytic enzyme preparations suitable for use in thepresent invention include, for example, SHEARZYME™ (Novozymes A/S),CELLIC™ HTec (Novozymes A/S), CELLIC™ HTec2 (Novozymes A/S), VISCOZYME®(Novozymes A/S), ULTRAFLO® (Novozymes A/S), PULPZYME® HC (NovozymesA/S), MULTIFECT® Xylanase (Genencor), ACCELLERASE® XY (Genencor),ACCELLERASE® XC (Genencor), ECOPULP® TX-200A (AB Enzymes), HSP 6000Xylanase (DSM), DEPOL™ 333P (Biocatalysts Limit, Wales, UK), DEPOL™740L. (Biocatalysts Limit, Wales, UK), and DEPOL™ 762P (BiocatalystsLimit, Wales, UK).

Examples of xylanases useful in the methods of the present inventioninclude, but are not limited to, Aspergillus aculeatus xylanase(GeneSeqP:AAR63790; WO 94/21785), Aspergillus fumigatus xylanases (WO2006/078256), and Thielavia terrestris NRRL 8126 xylanases (WO2009/079210).

Examples of beta-xylosidases useful in the methods of the presentinvention include, but are not limited to, Trichoderma reeseibeta-xylosidase (UniProtKB/TrEMBL accession number Q92458), Talaromycesemersonii (SwissProt accession number Q8X212), and Neurospora crassa(SwissProt accession number Q7SOW4).

Examples of acetylxylan esterases useful in the methods of the presentinvention include, but are not limited to, Hypocrea jecorina acetylxylanesterase (WO 2005/001036), Neurospora crassa acetylxylan esterase(UniProt accession number q7s259), Thielavia terrestris NRRL 8126acetylxylan esterase (WO 2009/042846), Chaetomium globosum acetylxylanesterase (Uniprot accession number Q2GWX4), Chaetomium gracileacetylxylan esterase (GeneSeqP accession number AAB82124), Phaeosphaerianodorum acetylxylan esterase (Uniprot accession number Q0UHJ1), andHumicola insolens DSM 1800 acetylxylan esterase (WO 2009/073709).

Examples of ferulic acid esterases useful in the methods of the presentinvention include, but are not limited to, Humicola insolens DSM 1800feruloyl esterase (WO 2009/076122), Neurospora crassa feruloyl esterase(UniProt accession number Q9HGR3), and Neosartorya fischeri feruloylesterase (UniProt Accession number A1D9T4).

Examples of arabinofuranosidases useful in the methods of the presentinvention include, but are not limited to, Humicola insolens DSM 1800arabinofuranosidase (WO 2009/073383) and Aspergillus nigerarabinofuranosidase (GeneSeqP accession number AAR94170).

Examples of alpha-glucuronidases useful in the methods of the presentinvention include, but are not limited to, Aspergillus clavatusalpha-glucuronidase (UniProt accession number alcc12), Trichodermareesei alpha-glucuronidase (Uniprot accession number Q99024),Talaromyces emersonii alpha-glucuronidase (UniProt accession numberQ8X211), Aspergillus niger alpha-glucuronidase (Uniprot accession numberQ96WX9), Aspergillus terreus alpha-glucuronidase (SwissProt accessionnumber Q0CJP9), and Aspergillus fumigatus alpha-glucuronidase (SwissProtaccession number Q4WW45).

The enzymes and proteins used in the methods of the present inventionmay be produced by fermentation of the above-noted microbial strains ona nutrient medium containing suitable carbon and nitrogen sources andinorganic salts, using procedures known in the art (see, e.g., Bennett,J. W. and LaSure, L. (eds.), More Gene Manipulations in Fungi, AcademicPress, Calif., 1991). Suitable media are available from commercialsuppliers or may be prepared according to published compositions (e.g.,in catalogues of the American Type Culture Collection). Temperatureranges and other conditions suitable for growth and enzyme productionare known in the art (see, e.g., Bailey, J. E., and Ollis, D. F.,Biochemical Engineering Fundamentals, McGraw-Hill Book Company, N.Y.,1986).

The fermentation can be any method of cultivation of a cell resulting inthe expression or isolation of an enzyme. Fermentation may, therefore,be understood as comprising shake flask cultivation, or small- orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing the enzymeto be expressed or isolated. The resulting enzymes produced by themethods described above may be recovered from the fermentation mediumand purified by conventional procedures.

Nucleic Acid Constructs

An isolated polynucleotide encoding a polypeptide, e.g., a polypeptidehaving cellulolytic enhancing activity, a cellulolytic enzyme, ahemicellulolytic enzyme, etc., may be manipulated in a variety of waysto provide for expression of the polypeptide by constructing a nucleicacid construct comprising an isolated polynucleotide encoding thepolypeptide operably linked to one or more (e.g., several) controlsequences that direct the expression of the coding sequence in asuitable host cell under conditions compatible with the controlsequences. Manipulation of the polynucleotide's sequence prior to itsinsertion into a vector may be desirable or necessary depending on theexpression vector. The techniques for modifying polynucleotide sequencesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter sequence, a polynucleotide thatis recognized by a host cell for expression of a polynucleotide encodinga polypeptide. The promoter sequence contains transcriptional controlsequences that mediate the expression of the polypeptide. The promotermay be any polynucleotide that shows transcriptional activity in thehost cell of choice including mutant, truncated, and hybrid promoters,and may be obtained from genes encoding extracellular or intracellularpolypeptides either homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs in the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes, E. colilac operon, Streptomyces coelicolor agarase gene (dagA), and prokaryoticbeta-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci.USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983,Proc. Natl. Acad. Sci. USA 80: 21-25). Further promoters are describedin “Useful proteins from recombinant bacteria” in Gilbert et al., 1980,Scientific American, 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs in the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase, aswell as the NA2-tpi promoter (a modified promoter from a gene encoding aneutral alpha-amylase in Aspergilli in which the untranslated leader hasbeen replaced by an untranslated leader from a gene encoding triosephosphate isomerase in Aspergilli; non-limiting examples includemodified promoters from the gene encoding neutral alpha-amylase inAspergillus niger in which the untranslated leader has been replaced byan untranslated leader from the gene encoding triose phosphate isomerasein Aspergillus nidulans or Aspergillus oryzae); and mutant, truncated,and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, which is recognized by a host cell to terminate transcription.The terminator sequence is operably linked to the 3′-terminus of thepolynucleotide encoding the polypeptide. Any terminator that isfunctional in the host cell of choice may be used in the presentinvention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase,Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, whentranscribed is a nontranslated region of an mRNA that is important fortranslation by the host cell. The leader sequence is operably linked tothe 5′-terminus of the polynucleotide encoding the polypeptide. Anyleader sequence that is functional in the host cell of choice may beused.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell of choice may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. The foreign signal peptide coding sequence may be requiredwhere the coding sequence does not naturally contain a signal peptidecoding sequence. Alternatively, the foreign signal peptide codingsequence may simply replace the natural signal peptide coding sequencein order to enhance secretion of the polypeptide. However, any signalpeptide coding sequence that directs the expressed polypeptide into thesecretory pathway of a host cell of choice may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present at theN-terminus of a polypeptide, the propeptide sequence is positioned nextto the N-terminus of a polypeptide and the signal peptide sequence ispositioned next to the N-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those that causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the Aspergillus niger glucoamylasepromoter, Aspergillus oryzae TAKA alpha-amylase promoter, andAspergillus oryzae glucoamylase promoter may be used. Other examples ofregulatory sequences are those that allow for gene amplification. Ineukaryotic systems, these regulatory sequences include the dihydrofolatereductase gene that is amplified in the presence of methotrexate, andthe metallothionein genes that are amplified with heavy metals. In thesecases, the polynucleotide encoding the polypeptide would be operablylinked with the regulatory sequence.

Expression Vectors

The various nucleotide and control sequences may be joined together toproduce a recombinant expression vector that may include one or more(e.g., several) convenient restriction sites to allow for insertion orsubstitution of a polynucleotide encoding a polypeptide, e.g., apolypeptide having cellulolytic enhancing activity, a cellulolyticenzyme, a hemicellulolytic enzyme, etc., at such sites. Alternatively,the polynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the sequence into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more (e.g., several) selectablemarkers that permit easy selection of transformed, transfected,transduced, or the like cells. A selectable marker is a gene the productof which provides for biocide or viral resistance, resistance to heavymetals, prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are the dal genes from Bacillussubtilis or Bacillus licheniformis, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, ortetracycline resistance. Suitable markers for yeast host cells are ADE2,HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMß1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide may be inserted into a host cellto increase production of a polypeptide. An increase in the copy numberof the polynucleotide can be obtained by integrating at least oneadditional copy of the sequence into the host cell genome or byincluding an amplifiable selectable marker gene with the polynucleotidewhere cells containing amplified copies of the selectable marker gene,and thereby additional copies of the polynucleotide, can be selected forby cultivating the cells in the presence of the appropriate selectableagent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors are well known to one skilled in theart (see, e.g., Sambrook et al., 1989, supra).

Host Cells

Recombinant host cells comprising a polynucleotide encoding apolypeptide, e.g., a polypeptide having cellulolytic enhancing activity,a cellulolytic enzyme, a hemicellulolytic enzyme, etc., can beadvantageously used in the recombinant production of the polypeptide. Aconstruct or vector comprising such a polynucleotide is introduced intoa host cell so that the vector is maintained as a chromosomal integrantor as a self-replicating extra-chromosomal vector as described earlier.The term “host cell” encompasses any progeny of a parent cell that isnot identical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the polypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any gram-positive or gram-negativebacterium. Gram-positive bacteria include, but not limited to, Bacillus,Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus,Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces.Gram-negative bacteria include, but not limited to, Campylobacter, E.coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter,Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell including, butnot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may, for instance, beeffected by protoplast transformation (see, e.g., Chang and Cohen, 1979,Mol. Gen. Genet. 168: 111-115), by using competent cells (see, e.g.,Young and Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), by electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or byconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may, forinstance, be effected by protoplast transformation (see, e.g., Hanahan,1983, J. Mol. Biol. 166: 557-580) or electroporation (see, e.g., Doweret al., 1988, Nucleic Acids Res. 16: 6127-6145). The introduction of DNAinto a Streptomyces cell may, for instance, be effected by protoplasttransformation and electroporation (see, e.g., Gong et al., 2004, FoliaMicrobiol. (Praha) 49: 399-405), by conjugation (see, e.g., Mazodier etal., 1989, J. Bacteriol. 171: 3583-3585), or by transduction (see, e.g.,Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98: 6289-6294). Theintroduction of DNA into a Pseudomonas cell may, for instance, beeffected by electroporation (see, e.g., Choi et al., 2006, J. Microbiol.Methods 64: 391-397) or by conjugation (see, e.g., Pinedo and Smets,2005, Appl. Environ. Microbiol. 71: 51-57). The introduction of DNA intoa Streptococcus cell may, for instance, be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), by protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), by electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or by conjugation(see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (asdefined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary ofThe Fungi, 8th edition, 1995, CAB International, University Press,Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al.,1995, supra, page 171) and all mitosporic fungi (Hawksworth et al.,1995, supra).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, F. A., Passmore, S. M., andDavenport, R. R., eds, Soc. App. Bacteriol. Symposium Series No. 9,1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phiebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

Methods for producing a polypeptide, e.g., a polypeptide havingcellulolytic enhancing activity, a cellulolytic enzyme, ahemicellulolytic enzyme, etc., comprise (a) cultivating a cell, which inits wild-type form is capable of producing the polypeptide, underconditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

Alternatively, methods for producing a polypeptide, e.g., a polypeptidehaving cellulolytic enhancing activity, a cellulolytic enzyme, ahemicellulolytic enzyme, etc., comprise (a) cultivating a recombinanthost cell under conditions conducive for production of the polypeptide;and (b) recovering the polypeptide.

In the production methods, the cells are cultivated in a nutrient mediumsuitable for production of the polypeptide using methods well known inthe art. For example, the cell may be cultivated by shake flaskcultivation, and small-scale or large-scale fermentation (includingcontinuous, batch, fed-batch, or solid state fermentations) inlaboratory or industrial fermentors performed in a suitable medium andunder conditions allowing the polypeptide to be expressed and/orisolated. The cultivation takes place in a suitable nutrient mediumcomprising carbon and nitrogen sources and inorganic salts, usingprocedures known in the art. Suitable media are available fromcommercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide. The polypeptides havingcellulolytic enhancing activity are detected using the methods describedherein.

The resulting broth may be used as is or the polypeptide may berecovered using methods known in the art. For example, the polypeptidemay be recovered from the nutrient medium by conventional proceduresincluding, but not limited to, centrifugation, filtration, extraction,spray-drying, evaporation, or precipitation.

The polypeptides may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Jansonand Lars Ryden, editors, VCH Publishers, New York, 1989) to obtainsubstantially pure polypeptides.

In an alternative aspect, the polypeptide is not recovered, but rather ahost cell expressing a polypeptide is used as a source of thepolypeptide.

Methods for Processing Cellulosic Material

The compositions and methods of the present invention can be used tosaccharify a cellulosic material to fermentable sugars and convert thefermentable sugars to many useful substances, e.g., fuel, potableethanol, and/or fermentation products (e.g., acids, alcohols, ketones,gases, and the like). The production of a desired fermentation productfrom cellulosic material typically involves pretreatment, enzymatichydrolysis (saccharification), and fermentation.

The present invention also relates to methods for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellulolytic enhancing activity and a liquor. In one aspect, themethod above further comprises recovering the degraded or convertedcellulosic material. Soluble products of degradation or conversion ofthe cellulosic material can be separated from the insoluble cellulosicmaterial using technology well known in the art such as, for example,centrifugation, filtration, and gravity settling.

The present invention also relates to methods for producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellulolytic enhancing activity and a liquor; (b) fermenting thesaccharified cellulosic material with one or more (e.g., several)fermenting microorganisms to produce the fermentation product; and (c)recovering the fermentation product from the fermentation.

The present invention also relates to methods of fermenting a cellulosicmaterial, comprising: fermenting the cellulosic material with one ormore (e.g., several) fermenting microorganisms, wherein the cellulosicmaterial is saccharified with an enzyme composition in the presence of apolypeptide having cellulolytic enhancing activity and a liquor. In oneaspect, the fermenting of the cellulosic material produces afermentation product. In another aspect, the method further comprisesrecovering the fermentation product from the fermentation.

In one aspect, the liquor is recovered following saccharification orfermentation and recycled back to a new saccharification reaction.Recycling of the liquor can be accomplished using processes conventionalin the art.

The processing of cellulosic material according to the present inventioncan be accomplished using processes conventional in the art. Moreover,the methods of the present invention can be implemented using anyconventional biomass processing apparatus configured to operate inaccordance with the invention.

Hydrolysis (saccharification) and fermentation, separate orsimultaneous, include, but are not limited to, separate hydrolysis andfermentation (SHF); simultaneous saccharification and fermentation(SSF); simultaneous saccharification and cofermentation (SSCF); hybridhydrolysis and fermentation (HHF); separate hydrolysis andco-fermentation (SHCF); hybrid hydrolysis and co-fermentation (HHCF);and direct microbial conversion (DMC), also sometimes calledconsolidated bioprocessing (CBP). SHF uses separate process steps tofirst enzymatically hydrolyze cellulosic material to fermentable sugars,e.g., glucose, cellobiose, cellotriose, and pentose monomers, and thenferment the fermentable sugars to ethanol. In SSF, the enzymatichydrolysis of cellulosic material and the fermentation of sugars toethanol are combined in one step (Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212). SSCF involves the cofermentation of multiple sugars (Sheehan,J., and Himmel, M., 1999, Enzymes, energy and the environment: Astrategic perspective on the U.S. Department of Energy's research anddevelopment activities for bioethanol, Biotechnol. Prog. 15: 817-827).HHF involves a separate hydrolysis step, and in addition a simultaneoussaccharification and hydrolysis step, which can be carried out in thesame reactor. The steps in an HHF process can be carried out atdifferent temperatures, i.e., high temperature enzymaticsaccharification followed by SSF at a lower temperature that thefermentation strain can tolerate. DMC combines all three processes(enzyme production, hydrolysis, and fermentation) in one or more (e.g.,several) steps where the same organism is used to produce the enzymesfor conversion of the cellulosic material to fermentable sugars and toconvert the fermentable sugars into a final product (Lynd, L. R.,Weimer, P. J., van Zyl, W. H., and Pretorius, I. S., 2002, Microbialcellulose utilization: Fundamentals and biotechnology, Microbiol. Mol.Biol. Reviews 66: 506-577). It is understood herein that any methodknown in the art comprising pretreatment, enzymatic hydrolysis(saccharification), fermentation, or a combination thereof, can be usedin the practicing the methods of the present invention.

A conventional apparatus can include a fed-batch stirred reactor, abatch stirred reactor, a continuous flow stirred reactor withultrafiltration, and/or a continuous plug-flow column reactor (Fernandade Castilhos Corazza, Flávio Faria de Moraes, Gisella Maria Zanin andIvo Neitzel, 2003, Optimal control in fed-batch reactor for thecellobiose hydrolysis, Acta Scientiarum. Technology 25: 33-38; Gusakov,A. V., and Sinitsyn, A. P., 1985, Kinetics of the enzymatic hydrolysisof cellulose: 1. A mathematical model for a batch reactor process, Enz.Microb. Technol. 7: 346-352), an attrition reactor (Ryu, S. K., and Lee,J. M., 1983, Bioconversion of waste cellulose by using an attritionbioreactor, Biotechnol. Bioeng. 25: 53-65), or a reactor with intensivestirring induced by an electromagnetic field (Gusakov, A. V., Sinitsyn,A. P., Davydkin, I. Y., Davydkin, V. Y., Protas, O. V., 1996,Enhancement of enzymatic cellulose hydrolysis using a novel type ofbioreactor with intensive stirring induced by electromagnetic field,Appl. Biochem. Biotechnol. 56: 141-153). Additional reactor typesinclude: fluidized bed, upflow blanket, immobilized, and extruder typereactors for hydrolysis and/or fermentation.

Pretreatment. In practicing the methods of the present invention, anypretreatment process known in the art can be used to disrupt plant cellwall components of cellulosic material (Chandra et al., 2007, Substratepretreatment: The key to effective enzymatic hydrolysis oflignocellulosics? Adv. Biochem. Engin./Biotechnol. 108: 67-93; Galbe andZacchi, 2007, Pretreatment of lignocellulosic materials for efficientbioethanol production, Adv. Biochem. Engin./Biotechnol. 108: 41-65;Hendriks and Zeeman, 2009, Pretreatments to enhance the digestibility oflignocellulosic biomass, Bioresource Technol. 100: 10-18; Mosier et al.,2005, Features of promising technologies for pretreatment oflignocellulosic biomass, Bioresource Technol. 96: 673-686; Taherzadehand Karimi, 2008, Pretreatment of lignocellulosic wastes to improveethanol and biogas production: A review, Int. J. of Mol. Sci. 9:1621-1651; Yang and Wyman, 2008, Pretreatment: the key to unlockinglow-cost cellulosic ethanol, Biofuels Bioproducts andBiorefining-Biofpr. 2: 26-40).

The cellulosic material can also be subjected to particle sizereduction, pre-soaking, wetting, washing, or conditioning prior topretreatment using methods known in the art.

Conventional pretreatments include, but are not limited to, steampretreatment (with or without explosion), dilute acid pretreatment, hotwater pretreatment, alkaline pretreatment, lime pretreatment, wetoxidation, wet explosion, ammonia fiber explosion, organosolvpretreatment, and biological pretreatment. Additional pretreatmentsinclude ammonia percolation, ultrasound, electroporation, microwave,supercritical CO₂, supercritical H₂O, ozone, and gamma irradiationpretreatments.

The cellulosic material can be pretreated before hydrolysis and/orfermentation. Pretreatment is preferably performed prior to thehydrolysis. Alternatively, the pretreatment can be carried outsimultaneously with enzyme hydrolysis to release fermentable sugars,such as glucose, xylose, and/or cellobiose. In most cases thepretreatment step itself results in some conversion of biomass tofermentable sugars (even in absence of enzymes).

Steam Pretreatment: In steam pretreatment, cellulosic material is heatedto disrupt the plant cell wall components, including lignin,hemicellulose, and cellulose to make the cellulose and other fractions,e.g., hemicellulose, accessible to enzymes. Cellulosic material ispassed to or through a reaction vessel where steam is injected toincrease the temperature to the required temperature and pressure and isretained therein for the desired reaction time. Steam pretreatment ispreferably done at 140-230° C., more preferably 160-200° C., and mostpreferably 170-190° C., where the optimal temperature range depends onany addition of a chemical catalyst. Residence time for the steampretreatment is preferably 1-15 minutes, more preferably 3-12 minutes,and most preferably 4-10 minutes, where the optimal residence timedepends on temperature range and any addition of a chemical catalyst.Steam pretreatment allows for relatively high solids loadings, so thatcellulosic material is generally only moist during the pretreatment. Thesteam pretreatment is often combined with an explosive discharge of thematerial after the pretreatment, which is known as steam explosion, thatis, rapid flashing to atmospheric pressure and turbulent flow of thematerial to increase the accessible surface area by fragmentation (Duffand Murray, 1996, Bioresource Technology 855: 1-33; Galbe and Zacchi,2002, Appl. Microbiol. Biotechnol. 59: 618-628; U.S. Patent ApplicationNo. 20020164730). During steam pretreatment, hemicellulose acetyl groupsare cleaved and the resulting acid autocatalyzes partial hydrolysis ofthe hemicellulose to monosaccharides and oligosaccharides. Lignin isremoved to only a limited extent.

A catalyst such as H₂SO₄ or SO₂ (typically 0.3 to 3% w/w) is often addedprior to steam pretreatment, which decreases the time and temperature,increases the recovery, and improves enzymatic hydrolysis (Ballesteroset al., 2006, Appl. Biochem. Biotechnol. 129-132: 496-508; Varga et al.,2004, Appl. Biochem. Biotechnol. 113-116: 509-523; Sassner et al., 2006,Enzyme Microb. Technol. 39: 756-762).

Chemical Pretreatment: The term “chemical treatment” refers to anychemical pretreatment that promotes the separation and/or release ofcellulose, hemicellulose, and/or lignin. Examples of suitable chemicalpretreatment processes include, for example, dilute acid pretreatment,lime pretreatment, wet oxidation, ammonia fiber/freeze explosion (AFEX),ammonia percolation (APR), and organosolv pretreatments.

In dilute acid pretreatment, cellulosic material is mixed with diluteacid, typically H₂SO₄, and water to form a slurry, heated by steam tothe desired temperature, and after a residence time flashed toatmospheric pressure. The dilute acid pretreatment can be performed witha number of reactor designs, e.g., plug-flow reactors, counter-currentreactors, or continuous counter-current shrinking bed reactors (Duff andMurray, 1996, supra; Schell et al., 2004, Bioresource Technol. 91:179-188; Lee et al., 1999, Adv. Biochem. Eng. Biotechnol. 65: 93-115).

Several methods of pretreatment under alkaline conditions can also beused. These alkaline pretreatments include, but are not limited to, limepretreatment, wet oxidation, ammonia percolation (APR), and ammoniafiber/freeze explosion (AFEX).

Lime pretreatment is performed with calcium carbonate, sodium hydroxide,or ammonia at low temperatures of 85-150° C. and residence times from 1hour to several days (Wyman et al., 2005, Bioresource Technol. 96:1959-1966; Mosier et al., 2005, Bioresource Technol. 96: 673-686). WO2006/110891, WO 2006/11899, WO 2006/11900, and WO 2006/110901 disclosepretreatment methods using ammonia.

Wet oxidation is a thermal pretreatment performed typically at 180-200°C. for 5-15 minutes with addition of an oxidative agent such as hydrogenperoxide or over-pressure of oxygen (Schmidt and Thomsen, 1998,Bioresource Technol. 64: 139-151; Palonen et al., 2004, Appl. Biochem.Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88:567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81:1669-1677). The pretreatment is performed at preferably 1-40% drymatter, more preferably 2-30% dry matter, and most preferably 5-20% drymatter, and often the initial pH is increased by the addition of alkalisuch as sodium carbonate.

A modification of the wet oxidation pretreatment method, known as wetexplosion (combination of wet oxidation and steam explosion), can handledry matter up to 30%. In wet explosion, the oxidizing agent isintroduced during pretreatment after a certain residence time. Thepretreatment is then ended by flashing to atmospheric pressure (WO2006/032282).

Ammonia fiber explosion (AFEX) involves treating cellulosic materialwith liquid or gaseous ammonia at moderate temperatures such as 90-100°C. and high pressure such as 17-20 bar for 5-10 minutes, where the drymatter content can be as high as 60% (Gollapalli et al., 2002, Appl.Biochem. Biotechnol. 98: 23-35; Chundawat et al., 2007, Biotechnol.Bioeng. 96: 219-231; Alizadeh et al., 2005, Appl. Biochem. Biotechnol.121: 1133-1141; Teymouri et al., 2005, Bioresource Technol. 96:2014-2018). AFEX pretreatment results in the depolymerization ofcellulose and partial hydrolysis of hemicellulose. Lignin-carbohydratecomplexes are cleaved.

Organosolv pretreatment delignifies cellulosic material by extractionusing aqueous ethanol (40-60% ethanol) at 160-200° C. for 30-60 minutes(Pan et al., 2005, Biotechnol. Bioeng. 90: 473-481; Pan et al., 2006,Biotechnol. Bioeng. 94: 851-861; Kurabi et al., 2005, Appl. Biochem.Biotechnol. 121: 219-230). Sulphuric acid is usually added as acatalyst. In organosolv pretreatment, the majority of hemicellulose isremoved.

Other examples of suitable pretreatment methods are described by Schellet al., 2003, Appl. Biochem. and Biotechnol. Vol. 105-108, p. 69-85, andMosier et al., 2005, Bioresource Technology 96: 673-686, and U.S.Published Application 2002/0164730.

In one aspect, the chemical pretreatment is preferably carried out as anacid treatment, and more preferably as a continuous dilute and/or mildacid treatment. The acid is typically sulfuric acid, but other acids canalso be used, such as acetic acid, citric acid, nitric acid, phosphoricacid, tartaric acid, succinic acid, hydrogen chloride, or mixturesthereof. Mild acid treatment is conducted in the pH range of preferably1-5, more preferably 1-4, and most preferably 1-3. In one aspect, theacid concentration is in the range from preferably 0.01 to 20 wt % acid,more preferably 0.05 to 10 wt % acid, even more preferably 0.1 to 5 wt %acid, and most preferably 0.2 to 2.0 wt % acid. The acid is contactedwith cellulosic material and held at a temperature in the range ofpreferably 160-220° C., and more preferably 165-195° C., for periodsranging from seconds to minutes to, e.g., 1 second to 60 minutes.

In another aspect, pretreatment is carried out as an ammonia fiberexplosion step (AFEX pretreatment step).

In another aspect, pretreatment takes place in an aqueous slurry. Inpreferred aspects, cellulosic material is present during pretreatment inamounts preferably between 10-80 wt %, more preferably between 20-70 wt%, and most preferably between 30-60 wt %, such as around 50 wt %. Thepretreated cellulosic material can be unwashed or washed using anymethod known in the art, e.g., washed with water.

Mechanical Pretreatment: The term “mechanical pretreatment” refers tovarious types of grinding or milling (e.g., dry milling, wet milling, orvibratory ball milling).

Physical Pretreatment: The term “physical pretreatment” refers to anypretreatment that promotes the separation and/or release of cellulose,hemicellulose, and/or lignin from cellulosic material. For example,physical pretreatment can involve irradiation (e.g., microwaveirradiation), steaming/steam explosion, hydrothermolysis, andcombinations thereof.

Physical pretreatment can involve high pressure and/or high temperature(steam explosion). In one aspect, high pressure means pressure in therange of preferably about 300 to about 600 psi, more preferably about350 to about 550 psi, and most preferably about 400 to about 500 psi,such as around 450 psi. In another aspect, high temperature meanstemperatures in the range of about 100 to about 300° C., preferablyabout 140 to about 235° C. In a preferred aspect, mechanicalpretreatment is performed in a batch-process, steam gun hydrolyzersystem that uses high pressure and high temperature as defined above,e.g., a Sunds Hydrolyzer available from Sunds Defibrator AB, Sweden.

Combined Physical and Chemical Pretreatment: Cellulosic material can bepretreated both physically and chemically. For instance, thepretreatment step can involve dilute or mild acid treatment and hightemperature and/or pressure treatment. The physical and chemicalpretreatments can be carried out sequentially or simultaneously, asdesired. A mechanical pretreatment can also be included.

Accordingly, in a preferred aspect, cellulosic material is subjected tomechanical, chemical, or physical pretreatment, or any combinationthereof, to promote the separation and/or release of cellulose,hemicellulose, and/or lignin.

Biological Pretreatment: The term “biological pretreatment” refers toany biological pretreatment that promotes the separation and/or releaseof cellulose, hemicellulose, and/or lignin from cellulosic material.Biological pretreatment techniques can involve applyinglignin-solubilizing microorganisms (see, for example, Hsu, T.-A., 1996,Pretreatment of biomass, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212; Ghosh and Singh, 1993, Physicochemical and biologicaltreatments for enzymatic/microbial conversion of cellulosic biomass,Adv. Appl. Microbiol. 39: 295-333; McMillan, J. D., 1994, Pretreatinglignocellulosic biomass: a review, in Enzymatic Conversion of Biomassfor Fuels Production, Himmel, M. E., Baker, J. O., and Overend, R. P.,eds., ACS Symposium Series 566, American Chemical Society, Washington,D.C., chapter 15; Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T.,1999, Ethanol production from renewable resources, in Advances inBiochemical Engineering/Biotechnology, Scheper, T., ed., Springer-VerlagBerlin Heidelberg, Germany, 65: 207-241; Olsson and Hahn-Hagerdal, 1996,Fermentation of lignocellulosic hydrolysates for ethanol production,Enz. Microb. Tech. 18: 312-331; and Vallander and Eriksson, 1990,Production of ethanol from lignocellulosic materials: State of the art,Adv. Biochem. Eng./Biotechnol. 42: 63-95).

Saccharification. In the hydrolysis step, also known assaccharification, the cellulosic material, e.g., pretreated, ishydrolyzed to break down cellulose and alternatively also hemicelluloseto fermentable sugars, such as glucose, cellobiose, xylose, xylulose,arabinose, mannose, galactose, and/or soluble oligosaccharides. Thehydrolysis is performed enzymatically by an enzyme composition in thepresence of a polypeptide having cellulolytic enhancing activity and aliquor. The enzyme and protein components of the compositions can beadded sequentially.

Enzymatic hydrolysis is preferably carried out in a suitable aqueousenvironment under conditions that can be readily determined by oneskilled in the art. In a preferred aspect, hydrolysis is performed underconditions suitable for the activity of the enzyme(s), i.e., optimal forthe enzyme(s). The hydrolysis can be carried out as a fed batch orcontinuous process where the pretreated cellulosic material (substrate)is fed gradually to, for example, an enzyme containing hydrolysissolution.

The saccharification is generally performed in stirred-tank reactors orfermentors under controlled pH, temperature, and mixing conditions.Suitable process time, temperature and pH conditions can readily bedetermined by one skilled in the art. For example, the saccharificationcan last up to 200 hours, but is typically performed for preferablyabout 12 to about 96 hours, more preferably about 16 to about 72 hours,and most preferably about 24 to about 48 hours. The temperature is inthe range of preferably about 25° C. to about 70° C., more preferablyabout 30° C. to about 65° C., and more preferably about 40° C. to 60°C., in particular about 50° C. The pH is in the range of preferablyabout 3 to about 8, more preferably about 3.5 to about 7, and mostpreferably about 4 to about 6, in particular about pH 5. The dry solidscontent is in the range of preferably about 5 to about 50 wt %, morepreferably about 10 to about 40 wt %, and most preferably about 20 toabout 30 wt %.

The optimum amounts of the enzymes and polypeptides having cellulolyticenhancing activity depend on several factors including, but not limitedto, the mixture of component cellulolytic enzymes, the cellulosicsubstrate, the concentration of cellulosic substrate, thepretreatment(s) of the cellulosic substrate, temperature, time, pH, andinclusion of fermenting organism (e.g., yeast for SimultaneousSaccharification and Fermentation).

In one aspect, an effective amount of cellulolytic or hemicellulolyticenzyme protein to cellulosic material is about 0.5 to about 50 mg,preferably at about 0.5 to about 40 mg, more preferably at about 0.5 toabout 25 mg, more preferably at about 0.75 to about 20 mg, morepreferably at about 0.75 to about 15 mg, even more preferably at about0.5 to about 10 mg, and most preferably at about 2.5 to about 10 mg perg of cellulosic material.

In another aspect, an effective amount of a polypeptide havingcellulolytic enhancing activity to cellulosic material is about 0.01 toabout 50.0 mg, preferably about 0.01 to about 40 mg, more preferablyabout 0.01 to about 30 mg, more preferably about 0.01 to about 20 mg,more preferably about 0.01 to about 10 mg, more preferably about 0.01 toabout 5 mg, more preferably at about 0.025 to about 1.5 mg, morepreferably at about 0.05 to about 1.25 mg, more preferably at about0.075 to about 1.25 mg, more preferably at about 0.1 to about 1.25 mg,even more preferably at about 0.15 to about 1.25 mg, and most preferablyat about 0.25 to about 1.0 mg per g of cellulosic material.

In another aspect, an effective amount of a polypeptide havingcellulolytic enhancing activity to cellulolytic enzyme protein is about0.005 to about 1.0 g, preferably at about 0.01 to about 1.0 g, morepreferably at about 0.15 to about 0.75 g, more preferably at about 0.15to about 0.5 g, more preferably at about 0.1 to about 0.5 g, even morepreferably at about 0.1 to about 0.5 g, and most preferably at about0.05 to about 0.2 g per g of cellulolytic enzyme protein.

Fermentation. The fermentable sugars obtained from the hydrolyzedcellulosic material can be fermented by one or more (e.g., several)fermenting microorganisms capable of fermenting the sugars directly orindirectly into a desired fermentation product. “Fermentation” or“fermentation process” refers to any fermentation process or any processcomprising a fermentation step. Fermentation processes also includefermentation processes used in the consumable alcohol industry (e.g.,beer and wine), dairy industry (e.g., fermented dairy products), leatherindustry, and tobacco industry. The fermentation conditions depend onthe desired fermentation product and fermenting organism and can easilybe determined by one skilled in the art.

In the fermentation step, sugars, released from cellulosic material as aresult of the pretreatment and enzymatic hydrolysis steps, are fermentedto a product, e.g., ethanol, by a fermenting organism, such as yeast.Hydrolysis (saccharification) and fermentation can be separate orsimultaneous, as described herein.

Any suitable hydrolyzed cellulosic material can be used in thefermentation step in practicing the present invention. The material isgenerally selected based on the desired fermentation product, i.e., thesubstance to be obtained from the fermentation, and the processemployed, as is well known in the art.

The term “fermentation medium” is understood herein to refer to a mediumbefore the fermenting microorganism(s) is(are) added, such as, a mediumresulting from a saccharification process, as well as a medium used in asimultaneous saccharification and fermentation process (SSF).

“Fermenting microorganism” refers to any microorganism, includingbacterial and fungal organisms, suitable for use in a desiredfermentation process to produce a fermentation product. The fermentingorganism can be C₆ and/or C₅ fermenting organisms, or a combinationthereof. Both C₆ and C₅ fermenting organisms are well known in the art.Suitable fermenting microorganisms are able to ferment, i.e., convert,sugars, such as glucose, xylose, xylulose, arabinose, maltose, mannose,galactose, or oligosaccharides, directly or indirectly into the desiredfermentation product.

Examples of bacterial and fungal fermenting organisms producing ethanolare described by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69:627-642.

Examples of fermenting microorganisms that can ferment C₆ sugars includebacterial and fungal organisms, such as yeast. Preferred yeast includesstrains of the Saccharomyces spp., preferably Saccharomyces cerevisiae.

Examples of fermenting organisms that can ferment C₅ sugars includebacterial and fungal organisms, such as some yeast. Preferred C₅fermenting yeast include strains of Pichia, preferably Pichia stipitis,such as Pichia stipitis CBS 5773; strains of Candida, preferably Candidaboidinii, Candida brassicae, Candida sheatae, Candida diddensii, Candidapseudotropicalis, or Candida utilis.

Other fermenting organisms include strains of Zymomonas, such asZymomonas mobilis; Hansenula, such as Hansenula anomala; Kluyveromyces,such as K. fragilis; Schizosaccharomyces, such as S. pombe; E. coli,especially E. coli strains that have been genetically modified toimprove the yield of ethanol; Clostridium, such as Clostridiumacetobutylicum, Chlostridium thermocellum, and Chlostridiumphytofermentans; Geobacillus sp.; Thermoanaerobacter, such asThermoanaerobacter saccharolyticum; and Bacillus, such as Bacilluscoagulans.

In a preferred aspect, the yeast is a Saccharomyces spp. In a morepreferred aspect, the yeast is Saccharomyces cerevisiae. In another morepreferred aspect, the yeast is Saccharomyces distaticus. In another morepreferred aspect, the yeast is Saccharomyces uvarum. In anotherpreferred aspect, the yeast is a Kluyveromyces. In another morepreferred aspect, the yeast is Kluyveromyces marxianus. In another morepreferred aspect, the yeast is Kluyveromyces fragilis. In anotherpreferred aspect, the yeast is a Candida. In another more preferredaspect, the yeast is Candida boidinii. In another more preferred aspect,the yeast is Candida brassicae. In another more preferred aspect, theyeast is Candida diddensii. In another more preferred aspect, the yeastis Candida pseudotropicalis. In another more preferred aspect, the yeastis Candida utilis. In another preferred aspect, the yeast is aClavispora. In another more preferred aspect, the yeast is Clavisporalusitaniae. In another more preferred aspect, the yeast is Clavisporaopuntiae. In another preferred aspect, the yeast is a Pachysolen. Inanother more preferred aspect, the yeast is Pachysolen tannophilus. Inanother preferred aspect, the yeast is a Pichia. In another morepreferred aspect, the yeast is a Pichia stipitis. In another preferredaspect, the yeast is a Bretannomyces. In another more preferred aspect,the yeast is Bretannomyces clausenii (Philippidis, G. P., 1996,Cellulose bioconversion technology, in Handbook on Bioethanol:Production and Utilization, Wyman, C. E., ed., Taylor & Francis,Washington, D.C., 179-212).

Bacteria that can efficiently ferment hexose and pentose to ethanolinclude, for example, Zymomonas mobilis, Clostridium acetobutylicum,Clostridium thermocellum, Chlostridium phytofermentans, Geobacillus sp.,Thermoanaerobacter saccharolyticum, and Bacillus coagulans (Philippidis,1996, supra).

In a preferred aspect, the bacterium is a Zymomonas. In a more preferredaspect, the bacterium is Zymomonas mobilis. In another preferred aspect,the bacterium is a Clostridium. In another more preferred aspect, thebacterium is Clostridium thermocellum.

Commercially available yeast suitable for ethanol production includes,e.g., ETHANOL RED™ yeast (available from Fermentis/Lesaffre, USA), FALI™(available from Fleischmann's Yeast, USA), SUPERSTART™ and THERMOSACC™fresh yeast (available from Ethanol Technology, Wis., USA), BIOFERM™ AFTand XR (available from NABC—North American Bioproducts Corporation, Ga.,USA), GERT STRAND™ (available from Gert Strand AB, Sweden), and FERMIOL™(available from DSM Specialties).

In a preferred aspect, the fermenting microorganism has been geneticallymodified to provide the ability to ferment pentose sugars, such asxylose utilizing, arabinose utilizing, and xylose and arabinoseco-utilizing microorganisms.

The cloning of heterologous genes into various fermenting microorganismshas led to the construction of organisms capable of converting hexosesand pentoses to ethanol (cofermentation) (Chen and Ho, 1993, Cloning andimproving the expression of Pichia stipitis xylose reductase gene inSaccharomyces cerevisiae, Appl. Biochem. Biotechnol. 39-40: 135-147; Hoet al., 1998, Genetically engineered Saccharomyces yeast capable ofeffectively cofermenting glucose and xylose, Appl. Environ. Microbiol.64: 1852-1859; Kotter and Ciriacy, 1993, Xylose fermentation bySaccharomyces cerevisiae, Appl. Microbiol. Biotechnol. 38: 776-783;Walfridsson et al., 1995, Xylose-metabolizing Saccharomyces cerevisiaestrains overexpressing the TKL1 and TAL1 genes encoding the pentosephosphate pathway enzymes transketolase and transaldolase, Appl.Environ. Microbiol. 61: 4184-4190; Kuyper et al., 2004, Minimalmetabolic engineering of Saccharomyces cerevisiae for efficientanaerobic xylose fermentation: a proof of principle, FEMS Yeast Research4: 655-664; Beall et al., 1991, Parametric studies of ethanol productionfrom xylose and other sugars by recombinant Escherichia coli, Biotech.Bioeng. 38: 296-303; Ingram et al., 1998, Metabolic engineering ofbacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214; Zhanget al., 1995, Metabolic engineering of a pentose metabolism pathway inethanologenic Zymomonas mobilis, Science 267: 240-243; Deanda et al.,1996, Development of an arabinose-fermenting Zymomonas mobilis strain bymetabolic pathway engineering, Appl. Environ. Microbiol. 62: 4465-4470;WO 2003/062430, xylose isomerase).

In a preferred aspect, the genetically modified fermenting microorganismis Saccharomyces cerevisiae. In another preferred aspect, thegenetically modified fermenting microorganism is Zymomonas mobilis. Inanother preferred aspect, the genetically modified fermentingmicroorganism is Escherichia coli. In another preferred aspect, thegenetically modified fermenting microorganism is Klebsiella oxytoca. Inanother preferred aspect, the genetically modified fermentingmicroorganism is Kluyveromyces sp.

It is well known in the art that the organisms described above can alsobe used to produce other substances, as described herein.

The fermenting microorganism is typically added to the degradedlignocellulose or hydrolysate and the fermentation is performed forabout 8 to about 96 hours, such as about 24 to about 60 hours. Thetemperature is typically between about 26° C. to about 60° C., inparticular about 32° C. or 50° C., and at about pH 3 to about pH 8, suchas around pH 4-5, 6, or 7.

In a preferred aspect, the yeast and/or another microorganism is appliedto the degraded cellulosic material and the fermentation is performedfor about 12 to about 96 hours, such as typically 24-60 hours. In apreferred aspect, the temperature is preferably between about 20° C. toabout 60° C., more preferably about 25° C. to about 50° C., and mostpreferably about 32° C. to about 50° C., in particular about 32° C. or50° C., and the pH is generally from about pH 3 to about pH 7,preferably around pH 4-7. However, some fermenting organisms, e.g.,bacteria, have higher fermentation temperature optima. Yeast or anothermicroorganism is preferably applied in amounts of approximately 10⁵ to10¹², preferably from approximately 10⁷ to 10¹⁰, especiallyapproximately 2×10⁸ viable cell count per ml of fermentation broth.Further guidance in respect of using yeast for fermentation can be foundin, e.g., “The Alcohol Textbook” (Editors K. Jacques, T. P. Lyons and D.R. Kelsall, Nottingham University Press, United Kingdom 1999), which ishereby incorporated by reference.

For ethanol production, following the fermentation the fermented slurryis distilled to extract the ethanol. The ethanol obtained according tothe methods of the invention can be used as, e.g., fuel ethanol,drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.

A fermentation stimulator can be used in combination with any of theprocesses described herein to further improve the fermentation process,and in particular, the performance of the fermenting microorganism, suchas, rate enhancement and ethanol yield. A “fermentation stimulator”refers to stimulators for growth of the fermenting microorganisms, inparticular, yeast. Preferred fermentation stimulators for growth includevitamins and minerals. Examples of vitamins include multivitamins,biotin, pantothenate, nicotinic acid, meso-inositol, thiamine,pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and VitaminsA, B, C, D, and E. See, for example, Alfenore et al., Improving ethanolproduction and viability of Saccharomyces cerevisiae by a vitaminfeeding strategy during fed-batch process, Springer-Verlag (2002), whichis hereby incorporated by reference. Examples of minerals includeminerals and mineral salts that can supply nutrients comprising P, K,Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation products: A fermentation product can be any substancederived from the fermentation. The fermentation product can be, withoutlimitation, an alcohol (e.g., arabinitol, n-butanol, isobutanol,ethanol, glycerol, methanol, ethylene glycol, 1,3-propanediol [propyleneglycol], butanediol, glycerin, sorbitol, and xylitol); an alkane (e.g.,pentane, hexane, heptane, octane, nonane, decane, undecane, anddodecane), a cycloalkane (e.g., cyclopentane, cyclohexane, cycloheptane,and cyclooctane), an alkene (e.g. pentene, hexene, heptene, and octene);an amino acid (e.g., aspartic acid, glutamic acid, glycine, lysine,serine, and threonine); a gas (e.g., methane, hydrogen (H₂), carbondioxide (CO₂), and carbon monoxide (CO)); isoprene; a ketone (e.g.,acetone); an organic acid (e.g., acetic acid, acetonic acid, adipicacid, ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid, formicacid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid,glutaric acid, 3-hydroxypropionic acid, itaconic acid, lactic acid,malic acid, malonic acid, oxalic acid, oxaloacetic acid, propionic acid,succinic acid, and xylonic acid); and polyketide. The fermentationproduct can also be protein as a high value product.

In a preferred aspect, the fermentation product is an alcohol. It willbe understood that the term “alcohol” encompasses a substance thatcontains one or more hydroxyl moieties. In a more preferred aspect, thealcohol is n-butanol. In another more preferred aspect, the alcohol isisobutanol. In another more preferred aspect, the alcohol is ethanol. Inanother more preferred aspect, the alcohol is methanol. In another morepreferred aspect, the alcohol is arabinitol. In another more preferredaspect, the alcohol is butanediol. In another more preferred aspect, thealcohol is ethylene glycol. In another more preferred aspect, thealcohol is glycerin. In another more preferred aspect, the alcohol isglycerol. In another more preferred aspect, the alcohol is1,3-propanediol. In another more preferred aspect, the alcohol issorbitol. In another more preferred aspect, the alcohol is xylitol. See,for example, Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T., 1999,Ethanol production from renewable resources, in Advances in BiochemicalEngineering/Biotechnology, Scheper, T., ed., Springer-Verlag BerlinHeidelberg, Germany, 65: 207-241; Silveira, M. M., and Jonas, R., 2002,The biotechnological production of sorbitol, Appl. Microbiol.Biotechnol. 59: 400-408; Nigam, P., and Singh, D., 1995, Processes forfermentative production of xylitol—a sugar substitute, ProcessBiochemistry 30 (2): 117-124; Ezeji, T. C., Qureshi, N. and Blaschek, H.P., 2003, Production of acetone, butanol and ethanol by Clostridiumbeijerinckii BA101 and in situ recovery by gas stripping, World Journalof Microbiology and Biotechnology 19 (6): 595-603.

In another preferred aspect, the fermentation product is an alkane. Thealkane can be an unbranched or a branched alkane. In another morepreferred aspect, the alkane is pentane. In another more preferredaspect, the alkane is hexane. In another more preferred aspect, thealkane is heptane. In another more preferred aspect, the alkane isoctane. In another more preferred aspect, the alkane is nonane. Inanother more preferred aspect, the alkane is decane. In another morepreferred aspect, the alkane is undecane. In another more preferredaspect, the alkane is dodecane.

In another preferred aspect, the fermentation product is a cycloalkane.In another more preferred aspect, the cycloalkane is cyclopentane. Inanother more preferred aspect, the cycloalkane is cyclohexane. Inanother more preferred aspect, the cycloalkane is cycloheptane. Inanother more preferred aspect, the cycloalkane is cyclooctane.

In another preferred aspect, the fermentation product is an alkene. Thealkene can be an unbranched or a branched alkene. In another morepreferred aspect, the alkene is pentene. In another more preferredaspect, the alkene is hexene. In another more preferred aspect, thealkene is heptene. In another more preferred aspect, the alkene isoctene.

In another preferred aspect, the fermentation product is an amino acid.In another more preferred aspect, the organic acid is aspartic acid. Inanother more preferred aspect, the amino acid is glutamic acid. Inanother more preferred aspect, the amino acid is glycine. In anothermore preferred aspect, the amino acid is lysine. In another morepreferred aspect, the amino acid is serine. In another more preferredaspect, the amino acid is threonine. See, for example, Richard, A., andMargaritis, A., 2004, Empirical modeling of batch fermentation kineticsfor poly(glutamic acid) production and other microbial biopolymers,Biotechnology and Bioengineering 87 (4): 501-515.

In another preferred aspect, the fermentation product is a gas. Inanother more preferred aspect, the gas is methane. In another morepreferred aspect, the gas is H₂. In another more preferred aspect, thegas is CO₂. In another more preferred aspect, the gas is CO. See, forexample, Kataoka, N., A. Miya, and K. Kiriyama, 1997, Studies onhydrogen production by continuous culture system of hydrogen-producinganaerobic bacteria, Water Science and Technology 36 (6-7): 41-47; andGunaseelan V. N. in Biomass and Bioenergy, Vol. 13 (1-2), pp. 83-114,1997, Anaerobic digestion of biomass for methane production: A review.

In another preferred aspect, the fermentation product is isoprene.

In another preferred aspect, the fermentation product is a ketone. Itwill be understood that the term “ketone” encompasses a substance thatcontains one or more ketone moieties. In another more preferred aspect,the ketone is acetone. See, for example, Qureshi and Blaschek, 2003,supra.

In another preferred aspect, the fermentation product is an organicacid. In another more preferred aspect, the organic acid is acetic acid.In another more preferred aspect, the organic acid is acetonic acid. Inanother more preferred aspect, the organic acid is adipic acid. Inanother more preferred aspect, the organic acid is ascorbic acid. Inanother more preferred aspect, the organic acid is citric acid. Inanother more preferred aspect, the organic acid is 2,5-diketo-D-gluconicacid. In another more preferred aspect, the organic acid is formic acid.In another more preferred aspect, the organic acid is fumaric acid. Inanother more preferred aspect, the organic acid is glucaric acid. Inanother more preferred aspect, the organic acid is gluconic acid. Inanother more preferred aspect, the organic acid is glucuronic acid. Inanother more preferred aspect, the organic acid is glutaric acid. Inanother preferred aspect, the organic acid is 3-hydroxypropionic acid.In another more preferred aspect, the organic acid is itaconic acid. Inanother more preferred aspect, the organic acid is lactic acid. Inanother more preferred aspect, the organic acid is malic acid. Inanother more preferred aspect, the organic acid is malonic acid. Inanother more preferred aspect, the organic acid is oxalic acid. Inanother more preferred aspect, the organic acid is propionic acid. Inanother more preferred aspect, the organic acid is succinic acid. Inanother more preferred aspect, the organic acid is xylonic acid. See,for example, Chen, R., and Lee, Y. Y., 1997, Membrane-mediatedextractive fermentation for lactic acid production from cellulosicbiomass, Appl. Biochem. Biotechnol. 63-65: 435-448.

In another preferred aspect, the fermentation product is polyketide.

Recovery. The fermentation product(s) can be optionally recovered fromthe fermentation medium using any method known in the art including, butnot limited to, chromatography, electrophoretic procedures, differentialsolubility, distillation, or extraction. For example, alcohol isseparated from the fermented cellulosic material and purified byconventional methods of distillation. Ethanol with a purity of up toabout 96 vol. % can be obtained, which can be used as, for example, fuelethanol, drinking ethanol, i.e., potable neutral spirits, or industrialethanol.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES

Media

2× YT plates were composed of 16 g of tryptone, 10 g of yeast extract, 5g of NaCl, 15 g of Noble agar, and deionized water to 1 liter.

PDA plates were composed of 39 grams of potato dextrose agar anddeionized water to 1 liter.

MDU2BP medium was composed of 45 g of maltose, 1 g of MgSO₄.7H₂O, 1 g ofNaCl, 2 g of K₂SO₄, 12 g of KH₂PO₄, 7 g of yeast extract, 2 g of urea,0.5 ml of AMG trace metals solution, and deionized water to 1 liter; pH5.0.

AMG trace metals solution was composed of 14.3 g of ZnSO₄.7H₂O, 2.5 g ofCuSO₄.5H₂O, 0.5 g of NiCl₂.6H₂O, 13.8 g of FeSO₄.7H₂O, 8.5 g ofMnSO₄.7H₂O, 3 g of citric acid, and deionized water to 1 liter.

Example 1 Pretreatment of Corn Stover

Corn stover was pretreated at the U.S. Department of Energy NationalRenewable Energy Laboratory (NREL) using 1.4% (w/v) sulfuric acid for 8minutes at 165° C. and 107 psi. The water-insoluble solids in thepretreated corn stover contained 57.5% cellulose, 4.6% hemicelluloses,and 28.4% lignin. Cellulose and hemicellulose were determined by atwo-stage sulfuric acid hydrolysis with subsequent analysis of sugars byhigh performance liquid chromatography using NREL Standard AnalyticalProcedure #002. Lignin was determined gravimetrically after hydrolyzingthe cellulose and hemicellulose fractions with sulfuric acid using NRELStandard Analytical Procedure #003.

The pretreated corn stover was adjusted to pH 5.0 by repeated additionof 10 N NaOH in aliquots of a few milliliters, followed by thoroughmixing and incubation at room temperature for approximately 1 hour. ThepH was confirmed after overnight incubation at 4° C., and thepH-adjusted corn stover was autoclaved for 20 minutes at approximately120° C., and then stored at 4° C. to minimize the risk of microbialcontamination. The dry weight of the pretreated corn stover was 33% TS(total solids), which was confirmed before each use.

The pretreated corn stover was milled prior to use. Milled pretreatedcorn stover (initial dry weight 32.35% TS) was prepared by milling in aCosmos ICMG 40 wet multi-utility grinder (EssEmm Corporation, TamilNadu, India). Milled pretreated corn stover was also, in some cases,subsequently washed repeatedly with deionized water followed bydecanting off the supernatant fraction. The dry weight of the milled,water-washed pretreated corn stover was 7.114% TS.

Alternatively, milled, water-washed pretreated corn stover was washedextensively with water at 50° C. Approximately 600 ml of water washedpretreated corn stover was diluted with approximately 500 ml ofdistilled, deionized water and incubated at 50° C. with shaking for 7days. Three to four times per day, the diluted pretreated corn stoverwas permitted to settle, and the supernatant water was decanted andreplaced with 500 ml of fresh deionized water. The dry weight of themilled, hot-water washed pretreated corn stover was 6.74% TS.

Example 2 Separation of Pretreated Corn Stover Liquor

Acid pretreated corn stover liquor was obtained by vacuum-filtration ofthe pH-adjusted NREL pretreated corn stover (Example 1) using Whatman #3filter paper in a Buchner funnel, or through a 0.22 μm STERICUP® sterilevacuum-filter (Millipore, Bedford, Mass., USA). For the Whatman-filteredliquor, the liquor was additionally sterile-filtered using a 0.22 μmSTERICUP® sterile vacuum-filter to minimize the risk of microbialcontamination.

In later experiments, pretreated corn stover liquor was obtained bysqueezing acid-pretreated corn stover in the following manner.Approximately 20 kg of dilute acid pretreated corn stover was loadedinto the cotton sheet lining of a SRL Water Press Model BP40-S/S(Zambelli Enotech, Camisano Vicentino, Italy). Municipal water pressure(approximately 35 psi) was applied for 20 minutes, and the resultingliquid pressed out was captured as liquor. This acidic liquor was storedat 4° C., and was subsequently pH-adjusted to 5.0 by addition of 10 NNaOH, and sterile-filtered using a 0.22 μm STERICUP® sterilevacuum-filter.

Example 3 Hydrolysis of Cellulose and Assay for GH61 PolypeptideEnhancement Thereof

The hydrolysis of pretreated corn stover was conducted using 2.2 ml,96-deep well plates (Axygen, Union City, Calif., USA) containing a totalreaction mass of 1 g. The hydrolysis was performed with 5% total solidsof either washed milled pretreated corn stover, unwashed pretreated cornstover, equivalent to 28.75 or 14.75 mg of cellulose per ml,respectively, or with a concentration of microcrystalline cellulose(AVICEL®, EM Science, Gibbstown, N.J., USA) equivalent to 28.75 mg ofcellulose per ml. Later hydrolysis reactions were performed with milled,washed or milled, unwashed pretreated corn stover with a cellulosecontent of 59%, equivalent to 29.5 mg of cellulose per ml, or anequivalent concentration of microcrystalline cellulose (AVICEL®,Sigma-Aldrich, St. Louis, Mo., USA). Hydrolysis reactions were performedin 50 mM sodium acetate pH 5.0 containing 1 mM manganese sulfate using aTrichoderma reesei cellulase preparation (CELLUCLAST® supplemented withAspergillus oryzae beta-glucosidase available from Novozymes A/S,Bagsvaerd, Denmark; the cellulase composition is designated herein inthe Examples as “Trichoderma reesei cellulase composition”) at 4 mg perg of cellulose. Thermoascus aurantiacus GH61A or Thelavia terrestrisGH61E polypeptide having cellulolytic enhancing activity was added atconcentrations between 0 and 50% (w/w) of total protein. Pretreated cornstover liquors, enzymatically- or chemically-treated corn stoverliquors, synthetic mixtures containing mono- and disaccharides atequivalent concentrations to corn stover liquors, pretreated biomasscomponent liquors, and post-fermentation residual liquors were addedbetween 0 and 20% (v/v) as indicated. Plates were sealed using anALPS-300™ plate heat sealer (Abgene, Epsom, United Kingdom) andincubated at 50° C. for 0-168 hours with mixing at 150 rpm. Allexperiments were performed in duplicate or triplicate. Other hydrolysisreactions were performed similarly, with the following differences:plates were sealed using an ALPS-3000™ plate heat sealer (Abgene, Epsom,United Kingdom), and incubated at 50° C. with vigorous initial mixing ateach sampling time, but mixing was not continuous.

At various time points between 24 and 168 hours of incubation, 100 μlaliquots were removed and the extent of hydrolysis was assayed byhigh-performance liquid chromatography (HPLC) using the protocoldescribed below.

For HPLC analysis, samples were filtered using a 0.45 μm MULTISCREEN®96-well filter plate (Millipore, Bedford, Mass., USA) and filtrates wereanalyzed for sugar content as described below. The sugar concentrationsof samples diluted in 0.005 M H₂SO₄ were measured using a 4.6×250 mmAMINEX® HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, Calif.,USA) by elution with 0.5% w/w benzoic acid-5 mM H₂SO₄ at a flow rate of0.6 ml per minute at 65° C. for 11 minutes, and quantification byintegration of glucose and cellobiose signals from refractive indexdetection (CHEMSTATION®, AGILENT® 1100 HPLC, Agilent Technologies, SantaClara, Calif., USA) calibrated by pure sugar samples. The resultantequivalents were used to calculate the fraction or percentage ofcellulose conversion for each reaction. The extent of each hydrolysiswas determined as the fraction of total cellulose converted tocellobiose+glucose, and was not corrected for soluble sugars present inpretreated corn stover liquor, or was corrected for soluble sugarspresent in liquor as indicated.

All HPLC data processing was performed using KALEIDAGRAPH® software(Synergy software, Reading, Pa., USA) or MICROSOFT EXCEL® (Microsoft,Seattle, Wash., USA). Measured sugar concentrations were adjusted forthe appropriate dilution factor. Glucose and cellobiose werechromatographically separated and integrated and their respectiveconcentrations determined independently. To calculate fractionalconversion the glucose and cellobiose values were combined. Fractionalhydrolysis is reported as the ratio of the mass corrected concentrationsof glucose and cellobiose to the initial concentration of cellulose asgiven by Equation 1. Triplicate data points were averaged and standarddeviation was calculated.

$\begin{matrix}{{{fractional}\mspace{14mu}{hydrolysis}} = \frac{\begin{matrix}\left( {\left( {\lbrack{cellobiose}\rbrack\left( {{mg}\text{/}{ml}} \right) \times 1.053} \right) +} \right. \\\left. {\left( {\lbrack{glucose}\rbrack\left( {{mg}\text{/}{ml}} \right)} \right)/1.111} \right)\end{matrix}}{\lbrack{cellulose}\rbrack\mspace{14mu}\left( {{mg}\text{/}{ml}} \right)}} & \left( {{Equation}\mspace{14mu} 1} \right)\end{matrix}$

The concentration-dependence of GH61 polypeptide-dependent enhancementof cellulose hydrolysis by the T. reesei cellulase composition wasdetermined by titration of the GH61 polypeptide between 0 and 50% (w/w)total protein added to a constant T. reesei cellulase concentration of 4mg per g cellulose, plotting fractional hydrolysis against GH61polypeptide concentration, and fitting using a modifiedsaturation-binding model as given by Equation 2.

$\begin{matrix}{{{fractional}\mspace{14mu}{hydrolysis}} = \frac{\begin{matrix}{{{\Delta fractional}\mspace{14mu}{hydrolysis} \times \left\lbrack {{GH}\; 61} \right\rbrack} +} \\{{fractional}\mspace{14mu}{{hydrolysis}_{(0)}\left( {K_{\frac{1}{2}{apparent}} + \left\lbrack {{GH}\; 61} \right\rbrack} \right)}}\end{matrix}}{K_{\frac{1}{2}{apparent}} + \left\lbrack {{GH}\; 61} \right\rbrack}} & \left( {{Equation}\mspace{14mu} 2} \right)\end{matrix}$In Equation 2 “fractional hydrolysis₍₀₎” was the hydrolysis in theabsence of a GH61 polypeptide; Δfractional hydrolysis was the total GH61polypeptide-dependent enhancement; i.e., the difference between thefractional hydrolysis at apparent “saturating” GH61 polypeptideconcentration and the fractional hydrolysis in the absence of the GH61polypeptide; and K_(1/2 apparent) was the GH61 polypeptide concentrationnecessary to observe a half-maximal enhancement of hydrolysis.

Example 4 Preparation of Thermoascus aurantiacus GH61A PolypeptideHaving Cellulolytic Enhancing Activity

Thermoascus aurantiacus GH61A polypeptide having cellulolytic enhancingactivity (SEQ ID NO: 13 [DNA sequence] and SEQ ID NO: 14 [deduced aminoacid sequence]) was recombinantly produced in Aspergillus oryzae JaL250according to WO 2005/074656. The recombinantly produced Thermoascusaurantiacus GH61A polypeptide was first concentrated by ultrafiltrationusing a 10 kDa membrane, buffer exchanged into 20 mM Tris-HCl pH 8.0,and then purified using a 100 ml Q-SEPHAROSE® Big Beads column (GEHealthcare, Piscataway, N.J., USA) with a 600 ml 0-600 mM NaCl lineargradient in the same buffer. Fractions of 10 ml were collected andpooled based on SDS-PAGE.

The pooled fractions (90 ml) were then further purified using a 20 mlMONO Q® column (GE Healthcare, Piscataway, N.J., USA) with a 500 ml0-500 mM NaCl linear gradient in the same buffer. Fractions of 6 ml werecollected and pooled based on SDS-PAGE. The pooled fractions (24 ml)were concentrated by ultrafiltration using a 10 kDa membrane, andchromatographed using a 320 ml SUPERDEX® 200 SEC column (GE Healthcare,Piscataway, N.J., USA) with isocratic elution of approximately 1.3 literof 150 mM NaCl-20 mM Tris-HCl pH 8.0. Fractions of 5 ml were collectedand pooled based on SDS-PAGE. Protein concentration was determined usinga Microplate BCA™ Protein Assay Kit (Thermo Fisher Scientific Inc.,Rockford, Ill., USA) in which bovine serum albumin was used as a proteinstandard.

Example 5 Preparation of Thielavia terrestris GH61E Polypeptide HavingCellulolytic Enhancing Activity

Thielavia terrestris GH61E polypeptide having cellulolytic enhancingactivity (SEQ ID NO: 7 [DNA sequence] and SEQ ID NO: 8 [deduced aminoacid sequence]) was recombinantly produced in Aspergillus oryzae JaL250according to U.S. Pat. No. 7,361,495. The Thielavia terrestris GH61Epolypeptide was desalted and buffer-exchanged into 20 mM sodiumacetate-150 mM NaCl pH 5.0 using a HIPREP® 26/10 desalting columnaccording to the manufacturer's instructions. Protein concentration wasdetermined using a Microplate BCA™ Protein Assay Kit in which bovineserum albumin was used as a protein standard.

Example 6 Preparation of Aspergillus fumigatus GH61B Polypeptide HavingCellulolytic Enhancing Activity

Aspergillus fumigatus GH61B polypeptide having cellulolytic enhancingactivity (SEQ ID NO: 29 [DNA sequence] and SEQ ID NO: 30 [deduced aminoacid sequence]) was recombinantly produced using Aspergillus oryzaeJaL355 as a host according to WO 2010/138754. The recombinantly producedA. fumigatus GH61B polypeptide was desalted and concentrated into 20 mMTris pH 8.0 using a 10 kDa MWCO membrane and purified by size exclusionchromatography using SUPERDEX® S75 (GE Healthcare, Piscataway, N.J.,USA). The purification buffer was 150 mM NaCl, 20 mM Tris 8.0.Homogeneity was confirmed by SDS-PAGE.

Example 7 Preparation of Penicillium pinophilum GH61A Polypeptide HavingCellulolytic Enhancing Activity

Penicillium pinophilum GH61A polypeptide having cellulolytic enhancingactivity (SEQ ID NO: 31 [DNA sequence] and SEQ ID NO: 32 [deduced aminoacid sequence]) was recombinantly produced using Aspergillus oryzaeHowB101 as a host according to WO 2011/005867. The recombinantlyproduced P. pinophilum GH61A polypeptide was desalted and concentratedinto 20 mM Tris pH 8.0 using a 10 kDa MWCO membrane and purified by sizeexclusion chromatography using SUPERDEX® S75. The purification bufferwas 150 mM NaCl, 20 mM Tris 8.0. Homogeneity was confirmed by SDS-PAGE.

Example 8 Preparation of Trichoderma reesei CEL7B Endoglucanase I

Trichoderma reesei CEL7B endoglucanase I (EGI) (SEQ ID NO: 65 [DNAsequence] and SEQ ID NO: 66 [deduced amino acid sequence]) was clonedand expressed in Aspergillus oryzae JaL250 as described in WO2005/067531. Filtered broth was concentrated and buffer exchanged usinga tangential flow concentrator equipped with a 10 kDa polyethersulfonemembrane with 20 mM Tris-HCl pH 8.5. The sample was loaded onto a QSEPHAROSE® High Performance column (GE Healthcare, Piscataway, N.J.,USA) equilibrated in 20 mM Tris pH 8.5, and bound proteins were elutedwith a linear gradient from 0-600 mM sodium chloride. The fractions wereconcentrated and desalted into 20 mM Tris pH 8.0, 150 mM NaCl usingVIVASPIN 20® 10 kDa MWCO centrifugal concentration devices (GEHealthcare UK limited, Little Chalfont, Buckinghamshire, UK). Proteinconcentration was determined using a Microplate BCA™ Protein Assay Kitin which bovine serum albumin was used as a protein standard.

Example 9 Preparation of Trichoderma reesei CEL5A Endoglucanase II

The Trichoderma reesei RutC30 Cel5A endoglucanase II gene (SEQ ID NO: 67[DNA sequence] and SEQ ID NO: 68 [deduced amino acid sequence]) wascloned and expressed in Aspergillus oryzae as described below.

Two synthetic oligonucleotide primers, shown below, were designed to PCRamplify the endoglucanase II gene from Trichoderma reesei RutC30 genomicDNA. Genomic DNA was isolated using a DNEASY® Plant Maxi Kit (QIAGENInc., Valencia, Calif., USA). An IN-FUSION™ PCR Cloning Kit (BDBiosciences, Palo Alto, Calif., USA) was used to clone the fragmentdirectly into pAlLo2 (WO 2004/099228).

Forward primer: (SEQ ID NO: 125)5′-ACTGGATTTACCATGAACAAGTCCGTGGCTCCATTGCT-3′ Reverse primer:(SEQ ID NO: 126) 5′-TCACCTCTAGTTAATTAACTACTTTCTTGCGAGACACG-3′Bold letters represent coding sequence. The remaining sequence containssequence identity to insertion sites of pAlLo2.

Fifty picomoles of each of the primers above were used in a PCR reactioncontaining 200 ng of Trichoderma reesei genomic DNA, 1× PfxAmplification Buffer (Invitrogen, Carlsbad, Calif., USA), 6 μl of 10 mMblend of dATP, dTTP, dGTP, and dCTP, 2.5 units of PLATINUM® Pfx DNApolymerase (Invitrogen Corp., Carlsbad, Calif., USA), and 1 μl of 50 mMMgSO₄ in a final volume of 50 μl. The amplification reaction wasincubated in an EPPENDORF® MASTERCYCLER® 5333 (Eppendorf Scientific,Inc., Westbury, N.Y., USA) programmed for one cycle at 98° C. for 2minutes; and 35 cycles each at 94° C. for 30 seconds, 61° C. for 30seconds, and 68° C. for 1.5 minutes. After the 35 cycles, the reactionwas incubated at 68° C. for 10 minutes and then cooled at 10° C. A 1.5kb PCR reaction product was isolated on a 0.8% GTG® agarose gel (CambrexBioproducts, East Rutherford, N.J., USA) using 40 mM Tris base, 20 mMsodium acetate, 1 mM disodium EDTA (TAE) buffer and 0.1 μg of ethidiumbromide per ml. The DNA band was visualized with the aid of aDARKREADER™ Transilluminator (Clare Chemical Research, Dolores, Colo.,USA). The 1.5 kb DNA band was excised with a disposable razor blade andpurified using an ULTRAFREE® DA spin cup (Millipore, Billerica, Mass.,USA) according to the manufacturer's instructions.

Plasmid pAlLo2 was linearized by digestion with Nco I and Pac I. Theplasmid fragment was purified by gel electrophoresis and ultrafiltrationas described above. Cloning of the purified PCR fragment into thelinearized and purified pAlLo2 vector was performed using an IN-FUSION™PCR Cloning Kit. The reaction (20 μl) contained 1× IN-FUSION™ Buffer (BDBiosciences, Palo Alto, Calif., USA), 1× BSA, 1 μl of IN-FUSION™ enzyme(diluted 1:10) (BD Biosciences, Palo Alto, Calif., USA), 100 ng ofpAlLo2 digested with Nco I and Pac I, and 100 ng of the Trichodermareesei Cel5A endoglucanase II PCR product. The reaction was incubated atroom temperature for 30 minutes. A 2 μl sample of the reaction was usedto transform E. coli XL10 SOLOPACK® Gold cells (Stratagene, La Jolla,Calif., USA) according to the manufacturer's instructions. After arecovery period, two 100 μl aliquots from the transformation reactionwere plated onto 150 mm 2× YT plates supplemented with 100 μg ofampicillin per ml. The plates were incubated overnight at 37° C. A setof 3 putative recombinant clones was recovered from the selection platesand plasmid DNA was prepared from each one using a BIOROBOT® 9600(QIAGEN Inc., Valencia, Calif., USA). Clones were analyzed by PciI/BspLU11 I restriction digestion. One clone with the expected restrictiondigestion pattern was then sequenced to confirm that there were nomutations in the cloned insert. Clone #3 was selected and designatedpAlLo27.

Aspergillus oryzae JaL250 protoplasts were prepared according to themethod of Christensen et al., 1988, supra. Five micrograms of pAlLo27(as well as pAlLo2 as a control) were used to transform Aspergillusoryzae JaL250 protoplasts. The transformation of Aspergillus oryzaeJaL250 with pAlLo27 yielded about 50 transformants. Eleven transformantswere isolated to individual PDA plates and incubated for five days at34° C.

Confluent spore plates were washed with 3 ml of 0.01% TWEEN® 80 (anonionic surfactant and emulsifier derived from polyethoxylated sorbitanand oleic acid) and the spore suspension was used to inoculate 25 ml ofMDU2BP medium in 125 ml glass shake flasks. Transformant cultures wereincubated at 34° C. with constant shaking at 200 rpm. At day fivepost-inoculation, cultures were centrifuged at 6000×g and theirsupernatants collected. Five microliters of each supernatant were mixedwith an equal volume of 2× loading buffer (10% beta-mercaptoethanol) andloaded onto a 1.5 mm 8%-16% Tris-glycine SDS-PAGE gel and stained withSIMPLYBLUE™ SafeStain (Invitrogen Corp., Carlsbad, Calif., USA).SDS-PAGE profiles of the culture broths showed that ten out of eleventransformants produced a new protein band of approximately 45 kDa.Transformant number 1, designated Aspergillus oryzae JaL250AlLo27, wascultivated in a fermentor.

One hundred ml of shake flask medium were added to a 500 ml shake flask.The shake flask medium was composed per liter of 50 g of sucrose, 10 gof KH₂PO₄, 0.5 g of CaCl₂, 2 g of MgSO₄.7H₂O, 2 g of K₂SO₄, 2 g of urea,10 g of yeast extract, 2 g of citric acid, and 0.5 ml of trace metalssolution. The trace metals solution was composed per liter of 13.8 g ofFeSO₄.7H₂O, 14.3 g of ZnSO₄.7H₂O, 8.5 g of MnSO₄.H₂O, 2.5 g ofCuSO₄.5H₂O, and 3 g of citric acid. The shake flask was inoculated withtwo plugs of Aspergillus oryzae JaL250AlLo27 from a PDA plate andincubated at 34° C. on an orbital shaker at 200 rpm for 24 hours. Fiftyml of the shake flask broth was used to inoculate a 3 liter fermentationvessel.

A total of 1.8 liters of the fermentation batch medium was added to athree liter glass jacketed fermentor (Applikon Biotechnology, Schiedam,Netherlands). The fermentation batch medium was composed per liter of 10g of yeast extract, 24 g of sucrose, 5 g of (NH₄)₂SO₄, 2 g of KH₂PO₄,0.5 g of CaCl₂.2H₂O, 2 g of MgSO₄.7H₂O, 1 g of citric acid, 2 g ofK₂SO₄, 0.5 ml of anti-foam, and 0.5 ml of trace metals solution. Thetrace metals solution was composed per liter of 13.8 g of FeSO₄.7H₂O,14.3 g of ZnSO₄.7H₂O, 8.5 g of MnSO₄.H₂O, 2.5 g of CuSO₄.5H₂O, and 3 gof citric acid. Fermentation feed medium was composed of maltose, whichwas dosed at a rate of 0 to 4.4 g/l/hr for a period of 185 hours. Thefermentation vessel was maintained at a temperature of 34° C. and pH wascontrolled using an Applikon 1030 control system (ApplikonBiotechnology, Schiedam, Netherlands) to a set-point of 6.1+/−0.1. Airwas added to the vessel at a rate of 1 vvm and the broth was agitated bya Rushton impeller rotating at 1100 to 1300 rpm. At the end of thefermentation, whole broth was harvested from the vessel and centrifugedat 3000×g to remove the biomass. The supernatant was sterile filteredand stored at 5 to 10° C.

The supernatant was desalted and buffer-exchanged into 20 mM Bis-Tris pH6.0 using a HIPREP® 26/10 desalting column (GE Healthcare, Piscataway,N.J., USA) according to the manufacturer's instructions. The bufferexchanged sample was loaded onto a MonoQ® column (GE Healthcare,Piscataway, N.J., USA) equilibrated with 20 mM Bis-Tris pH 6.0, and thebound protein was eluted with a linear gradient from 0 to 1000 mM sodiumchloride. Protein fractions were pooled and buffer exchanged into 1.2 M(NH₄)₂SO₄-20 mM Tris-HCl pH 8.5. The sample was loaded onto a PhenylSUPEROSE™ column (HR 16/10) equilibrated with 1.2 M (NH₄)₂SO₄-20 mMTris-HCl pH 8.0. Bound proteins were eluted with a linear gradient over20 column volumes from 1.2 to 0 M (NH₄)₂SO₄ in 20 mM Tris-HCl pH 8.5.The fractions were pooled, concentrated, and loaded onto a SUPERDEX® 75HR 26/60 column (GE Healthcare, Piscataway, N.J., USA) equilibrated with20 mM Tris-150 mM sodium chloride pH 8.5. Fractions were pooled andconcentrated in 20 mM Tris-150 mM sodium chloride pH 8.5. Proteinconcentration was determined using a Microplate BCA™ Protein Assay Kitin which bovine serum albumin was used as a protein standard.

Example 10 Preparation of Trichoderma reesei CEL7A Cellobiohydrolase I

Trichoderma reesei CEL7A cellobiohydrolase I (SEQ ID NO: 95 [DNAsequence] and SEQ ID NO: 96 [deduced amino acid sequence]) was preparedas described by Ding and Xu, 2004, “Productive cellulase adsorption oncellulose” in Lignocellulose Biodegradation (Saha, B. C. ed.), SymposiumSeries 889, pp.154-169, American Chemical Society, Washington, D.C.Protein concentration was determined using a Microplate BCA™ ProteinAssay Kit in which bovine serum albumin was used as a protein standard.

Example 11 Preparation of Trichoderma reesei CEL6A Cellobiohydrolase II

The Trichoderma reesei RutC30 CEL6A cellobiohydrolase II gene (SEQ IDNO: 97 [DNA sequence] and SEQ ID NO: 98 [deduced amino acid sequence])was isolated from Trichoderma reesei RutC30 as described in WO2005/056772. The Trichoderma reesei CEL6A cellobiohydrolase II gene wasexpressed in Fusarium venenatum using pEJG61 as an expression vectoraccording to the procedures described in U.S. Published Application No.20060156437. Fermentation was performed as described in U.S. PublishedApplication No. 20060156437. Filtered broth was desalted andbuffer-exchanged into 20 mM sodium acetate-150 mM NaCl pH 5.0 using aHIPREP® 26/10 Desalting Column according to the manufacturer'sinstructions. Protein concentration was determined using a MicroplateBCA™ Protein Assay Kit in which bovine serum albumin was used as aprotein standard.

Example 12 Preparation of Aspergillus oryzae CEL3A Beta-Glucosidase

Aspergillus oryzae CEL3A beta-glucosidase (SEQ ID NO: 111 [DNA sequence]and SEQ ID NO: 112 [deduced amino acid sequence]) was recombinantlyprepared as described in WO 2004/099228, and purified as described byLangston et al., 2006, Biochim. Biophys. Acta Proteins Proteomics 1764:972-978. Protein concentration was determined using a Microplate BCA™Protein Assay Kit.

Example 13 Effect of GH61 Polypeptides Having Cellulolytic EnhancingActivity on Hydrolysis of Microcrystalline Cellulose or PCS by theTrichoderma reesei Cellulase Composition

The effect of the Thermoascus aurantiacus GH61A polypeptide on thehydrolysis of AVICEL® or milled washed PCS by the Trichoderma reeseicellulase composition was determined using the same experimentalconditions and procedures according to Example 3. In general, inexperiments performed in subsequent examples, a control reaction wasincluded in which increasing concentrations of GH61 polypeptide wereadded to the hydrolysis of either AVICEL® or pretreated corn stover withthe T. reesei cellulase composition in the absence of other liquors,compounds, or variously treated liquors.

The presence of the T. aurantiacus GH61A polypeptide did not enhance thehydrolysis of AVICEL® by the T. reesei cellulase composition. Conversionof AVICEL® was 0.119±0.00251 and 0.339±0.00222 at 1 and 3 days,respectively, in the absence of the T. aurantiacus GH61A polypeptidecompared to 0.112±0.00376 and 0.333±0.00328 at 1 and 3, respectively, inthe presence of 24% (w/w) of the T. aurantiacus GH61A polypeptide.

The presence of the T. aurantiacus GH61A polypeptide enhanced thehydrolysis of milled washed PCS by the T. reesei cellulase composition.Conversion of milled washed PCS was 0.249±0.00104 and 0.545±0.00656 at 1and 3 days, respectively, in the presence of the T. aurantiacus GH61Apolypeptide compared to 0.222±0.00464 and 0.412±0.0237 at 1 and 3 days,respectively, in the absence of the T. aurantiacus GH61A polypeptide.

The presence of the T. aurantiacus GH61A polypeptide marginally enhancedthe hydrolysis of milled hot-washed PCS by the T. reesei cellulasecomposition. Conversion of hot washed PCS was 0.315±0.00267 and0.383±0.00498, at 1 and 3 days, respectively, in the absence of the T.aurantiacus GH61A polypeptide compared to 0.331±0.0115 and 0.409±0.0145at 1 and 3, respectively, in the presence of 8% (w/w) of the T.aurantiacus GH61A polypeptide.

The presence of the Thelavia terrestris GH61E polypeptide did notenhance the hydrolysis of AVICEL® by the T. reesei cellulasecomposition. Conversion of AVICEL® was 0.122±0.00426, 0.242±0.00813 and0.315±0.00814, at 1, 3, and 5 days, respectively, in the absence of theT. terrestris GH61E polypeptide compared with 0.121±0.000824,0.228±0.000978 and 0.307±0.00348 at 1, 3, and 5 days respectively, inthe presence of 24% (w/w) of the T. terrestris GH61E polypeptide.

The presence of the Aspergillus fumigatus GH61B polypeptide did notsignificantly enhance the hydrolysis of AVICEL® by the T. reeseicellulase composition. Conversion of AVICEL® was 0.150±0.009,0.31±0.001, and 0.48±0.001 at 1, 3, and 7 days, respectively, in theabsence of the A. fumigatus GH61B polypeptide compared to 0.148±0.002%,0.311±0.001%, and 0.54±0.02% at 1, 3, and 7 days, respectively, in thepresence of the A. fumigatus GH61B polypeptide.

Example 14 Effect of Thermoascus aurantiacus GH61A Polypeptide onCellulolysis of Unwashed and Washed Acid-Pretreated Corn Stover

The effect of Thermoascus aurantiacus GH61A polypeptide on hydrolysis ofboth milled washed and milled unwashed NREL pretreated corn stover bythe Trichoderma reesei cellulase composition were assayed forcomparison. To an equivalent, fixed concentration of the T. reeseicellulase composition at 4 mg per gram of cellulose, increasingconcentrations of T. aurantiacus GH61A polypeptide between 0 and 24%(w/w) were added to equivalent dry masses of milled unwashed pretreatedcorn stover or milled washed pretreated corn stover, and the fractionalhydrolysis was assayed according to Example 3.

FIG. 1 shows the fractional hydrolysis of variously washed pretreatedcorn stover substrates. The total enhancement in total conversion fromthe T. aurantiacus GH61A polypeptide was larger in magnitude and wasapparent at earlier stages of hydrolysis for unwashed corn stover incomparison to washed pretreated corn stover. Over the range ofconcentrations tested, there was no GH61 polypeptideconcentration-dependence of hydrolysis after 1 day of hydrolysis forwashed milled pretreated corn stover (open squares), whereas a slightbut significant increase of hydrolysis as GH61 polypeptide concentrationincreased was observed for unwashed milled corn stover (open circles).After 3 days of hydrolysis, washed pretreated corn stover showed asharp, saturable enhancement of hydrolysis with GH61 polypeptideaddition, well-fitted by a square hyperbolic binding function (closedsquares). Conversely, there was a linear increase in hydrolysis withGH61 polypeptide concentration for the unwashed pretreated corn stoverthat extrapolated to a higher overall conversion at GH61 polypeptideconcentrations greater than 50% (w/w) (closed circles). For hot-waterwashed pretreated corn stover prepared according to Example 1, thistrend was even more apparent; after 3 days of hydrolysis, no GH61polypeptide enhancement was observed (open triangles), and GH61polypeptide-dependent enhancement of cellulolysis was not apparent until5 days of hydrolysis (closed triangles). The total magnitude of GH61polypeptide-dependent enhancement for hot-water washed pretreated cornstover was only 5% at 7 days, whereas for the GH61 polypeptideenhancement for unwashed pretreated corn stover was at least 15% at 7days of hydrolysis (data not shown). In each case, the conversion by theT. reesei cellulase composition in the absence of the GH61 polypeptide(y-intercept) was higher for the more extensively washed substrates,indicating the removal of soluble cellulase inhibitors by washing.

Example 15 The Effect of Addition of Acid-Pretreated Corn Stover Liquorto Washed Milled Pretreated Corn Stover

Acid-pretreated corn stover liquor, fractionated according to Example 2,was added at concentrations between 0 and 15% (v/v) to milled,water-washed acid-pretreated corn stover and hydrolyzed by 4 mg of theTrichoderma reesei cellulase composition per g cellulose plus increasingconcentrations of the Thermoascus aurantiacus GH61A polypeptideaccording to Example 3.

FIG. 2A shows the extent of hydrolysis for the various additions of theT. aurantiacus GH61A polypeptide and NREL acid-pretreated corn stoverliquor to milled, water-washed acid-pretreated corn stover at 1 day(white bars) and 3 days (gray bars) of hydrolysis.

FIG. 2B shows a replot of the data presented in FIG. 2A with non-linearleast square fits to Equation 2 according to Example 3 or with linearleast square fits. The Figure demonstrates that the functionaldependence of the extent of hydrolysis was square hyperbolic, i.e.,saturating, for concentrations of liquor <10% (v/v), and became linearor exponential at concentrations of liquor ≥10% (v/v) at 3 days ofhydrolysis. The extent of hydrolysis at the highest added concentrationof the T. aurantiacus GH61A polypeptide was greater than that observedin the absence of liquor, and extrapolation of the trends to predictfractional hydrolysis levels at higher GH61 polypeptide concentrationsindicated a greater conversion at higher liquor concentrations. Fromfits of Equation 2, the total enhancement from the GH61 polypeptide,fractional hydrolysis, was 0.150±0.000550 in the absence of addedliquor, 0.166±0.0155 with 2% liquor, 0.227±0.0827 with 5% liquor, and0.417±0.240 with 10% liquor, and the increase was linear with GH61polypeptide concentration at 15% liquor.

Example 16 Effect of Addition of Acid-Pretreated Corn Stover Liquor onThermoascus aurantiacus GH61A Polypeptide During Hydrolysis ofMicrocrystalline Cellulose

Pretreated corn stover liquor, fractionated according to Example 2, wasadded to a saccharification reaction of microcrystalline cellulose bythe Trichoderma reesei cellulase composition according to Example 3 atconcentrations between 0 and 20% (v/v). The Thermoascus aurantiacusGH61A polypeptide was titrated between 0 and 24% (w/w) of total protein.

FIG. 3 shows that the T. aurantiacus GH61A polypeptide did not enhanceon hydrolysis of microcrystalline cellulose by the T. reesei cellulasecomposition in the absence of liquor. However, in the presence of NRELpretreated corn stover liquor, the T. aurantiacus GH61A polypeptideenhanced cellulolysis. FIG. 3A shows fractional hydrolysis at variousGH61 polypeptide concentrations for increasing concentrations of NRELpretreated corn stover liquor at 1 day (open circles) and 3 days ofhydrolysis (closed circles). As pretreated corn stover liquor was addedfrom 0% v/v (circles) to 5% (diamonds), 10% (triangles), and 15%(inverted triangles), in the absence of the T. aurantiacus GH61Apolypeptide, there was increasing inhibition of the T. reesei cellulasecomposition, as was apparent from the reduction in fractionalhydrolysis. As the concentration of the T. aurantiacus GH61A polypeptidewas increased, for those samples containing acid-pretreated corn stoverliquor, the extent of hydrolysis increased to an extent beyond the levelobserved for hydrolysis in the absence of liquor. These data werecorrected for saccharides present in the added liquor, and thepossibility that either the T. aurantiacus GH61A polypeptide or the T.reesei cellulase composition in combination with the T. aurantiacusGH61A polypeptide were converting some substrate in pretreated cornstover liquor to glucose was unlikely, as the extent of enhancement fromthe T. aurantiacus GH61A polypeptide at 5% liquor addition wouldcorrespond to a conversion of 55 g/L glucose equivalents from the addedliquor.

FIG. 3B shows the results of addition of a synthetic mixture of themajor sugar components of pretreated corn stover liquor; glucose,cellobiose, xylose, and arabinose with or without added phenol to mimicthe phenolic lignin degradation compounds present in pretreated cornstover liquor, at the concentrations present in actual liquor. Thesynthetic sugar mixture was added between 0% and 15%, (symbols as inFIG. 3A), and the synthetic sugar mixture containing phenol was added ateither 5% (squares) or 15% (right triangles). For this example, addedliquor sugar concentrations were not subtracted from the overallapparent hydrolysis, as was evident from the upward shift in apparenthydrolysis with increasing liquor addition. From FIG. 3B, fractionalhydrolysis was independent of GH61 polypeptide concentration, thus therewas no apparent GH61 polypeptide cellulolytic enhancing activity in thepresence of the synthetic liquor mixtures on microcrystalline cellulose.These data indicated that the GH61 polypeptide cellulolytic enhancingactivity derives from the minor components of NREL pretreated cornstover liquor.

Example 17 Effect of Preconditioning Acid-Pretreated Corn Stover Liquorwith Thelavia terrestris GH61E Polypeptide

Pretreated corn stover liquor, extracted according to Example 2, wasadded to a saccharification reaction of microcrystalline cellulose bythe Trichoderma reesei cellulase composition according to Example 3 atconcentrations between 0 and 20% (v/v). Alternatively, 10 ml of liquorwere incubated at 50° C. overnight with 0.43 mg protein of the T. reeseicellulase composition per ml, or with 20 μg protein of the Thielaviaterrestris GH61E polypeptide per ml, or both (pre-conditioned liquor).The enzyme was removed from these samples using a 3 kDa MWCO AMICON®centrifuge filter (Millipore, Bedford, Mass., USA), and the filteredflow-through liquor was added to saccharifications of microcrystallinecellulose at concentrations between 0 and 20% (v/v). The T. terrestrisGH61E polypeptide was titrated between concentrations of 0 and 24% (w/w)of total protein.

FIG. 4 shows that, like the T. aurantiacus GH61A polypeptidedemonstrated in Example 11, the Thelavia terrestris GH61E polypeptidehad cellulolytic enhancing activity on microcrystalline cellulose in thepresence of NREL pretreated corn stover liquor and did not enhancecellulolysis in the absence of liquor. FIG. 4 (all panels, circles)shows that increasing T. terrestris GH61E polypeptide concentration onmicrocrystalline cellulose in the absence of liquor did not enhancehydrolysis. As liquor was added from 0% (v/v) (circles) to 5% (squares),10% (diamonds), and 15% (triangles) in the absence of the T. terrestrisGH61E polypeptide, there was increasing inhibition of the T. reeseicellulase composition, as was apparent from the reduction in fractionalhydrolysis (all panels, y-intercepts). As the T. terrestris GH61Epolypeptide concentration was increased, for those samples containingeither liquor or enzymatically pre-conditioned liquor, the extent ofhydrolysis increased. In most cases, despite the inhibition ofcellulolysis arising from the liquor addition, the increase inhydrolysis led to an extent of saccharification beyond the levelobserved for hydrolysis in the absence of liquor at high Thelaviaterrestris GH61E polypeptide concentrations. FIG. 4A shows the effectsof liquor that had not been enzymatically treated, FIG. 4B shows theeffects of liquor that had been saccharified using the T. reeseicellulase composition, FIG. 4C shows the effects of liquor that had beenincubated with both the T. reesei cellulase composition and the T.terrestris GH61E polypeptide. In each case, GH61 polypeptidecellulolytic enhancing activity was apparent in the presence ofpretreated corn stover liquor. Control reactions containing identicalconcentrations of liquor or pretreated liquor and enzyme in identicalconcentrations to those present in the cellulose-containing samples wereperformed in parallel, and sugars produced from these control reactionswere subtracted from the saccharification totals. In each case theconversion of pretreated corn stover liquor to glucose equivalentsproduced minimal total sugar, and far less than was necessary to accountfor the large cellulolytic enhancement observed in saccharificationscontaining both the Thielavia terrestris GH61E polypeptide and liquor.

Example 18 Effect of Acid-Pretreated Corn Stover Liquor orSteam-Pretreated Corn Stover Liquor on Thelavia terrestris GH61EPolypeptide Activity

Pretreated corn stover liquor was extracted according to Example 2 withthe following exceptions: 0.6 kg of steam explosion-pretreated cornstover or NREL acid-pretreated corn stover were suspended in 900 ml ofdeionized water and mixed for 2 hours. The solids were filtered usingfilter paper and sterile filtered with a 0.45 μm filter. Two hundred mlof each was ultracentrifuged using a 44.5 mM diameter 1 kDa MWCOcentrifuge filter (Millipore, Bedford, Mass., USA). The concentratedretentate was restored to the original volume by addition of water. Theoriginal liquors and the molecular weight separated fractions of eachwere added to saccharification reactions of microcrystalline cellulosewith the Trichoderma reesei cellulase composition according to Example 3at concentrations of 5% and 15% (v/v). The Thelavia terrestris GH61Epolypeptide was titrated at concentrations between 0 and 24% (w/w) totalprotein.

FIG. 5A shows that increasing concentrations of the Thelavia terrestrisGH61E polypeptide increased the hydrolysis of microcrystalline celluloseby the T. reesei cellulase composition in the presence of theacid-pretreated corn stover liquor. Thus in the presence of theacid-pretreated corn stover liquor, the T. terrestris GH61E polypeptidesignificantly enhanced cellulolysis. FIG. 5B shows that increasingconcentrations of the Thelavia terrestris GH61E polypeptide marginallyincreased hydrolysis in the presence of steam explosion-pretreated cornstover, thus demonstrating minimal enhancement of cellulolysis byThelavia terrestris GH61E under similar conditions.

Overall, the results indicated that conditions of pretreatment arecritical for the production of liquor components necessary for GH61cellulolytic enhancing activity on microcrystalline cellulose. Moresevere pretreatments, exemplified herein by the acid-pretreated cornstover (e.g., NREL PCS), generate soluble components or higherconcentrations of the soluble components than do milder pretreatments,as illustrated by steam-pretreated corn stover above.

Example 19 Separation of GH61 Polypeptide-Enhancing Liquor ComponentsFrom Non-Enhancing Components and Inhibitory Components in NRELPretreated Corn Stover Liquor

Pretreated corn stover liquor was extracted and separated byultrafiltration into nominal molecular weight fractions above and below1 kDa according to Example 18. The molecular weight separated fractionswere added to a saccharification reaction of microcrystalline cellulosewith the T. reesei cellulase composition at concentrations between 0 and15% (v/v) according to Example 3. The Thelavia terrestris GH61Epolypeptide was titrated at concentrations between 0 and 24% (w/w) totalprotein.

As shown in FIG. 5A, at zero T. terrestris GH61E polypeptideconcentration, both the whole liquor and the lower molecular weightfractions showed inhibition at higher liquor concentrations (circles,squares). The inhibition from liquor has been discussed previously inExamples 10, 12 and 13. Conversely, there was no difference inhydrolysis at either 5 or 15% (v/v) of the higher molecular weightfraction (triangles). Increasing concentrations of the T. terrestrisGH61E polypeptide with all these liquor molecular weight fractionsincreased cellulose hydrolysis. The unfractionated pretreated cornstover liquor yielded the highest GH61 polypeptide-dependentenhancement, however the high molecular weight pretreated corn stoverliquor fraction also yielded substantial GH61 polypeptide-dependentenhancement.

The efficacy of molecular weight-based filtration was confirmed byfractionation of PCS liquor using AMICON® 30, 10, and 3 kDa MWCOcentrifuge filters (Millipore, Bedford, Mass., USA) and assaying forGH61 cellulolytic enhancing activity. NREL acid-pretreated corn stoverliquor was extracted according to Example 2, and then filtered throughsuccessively smaller MWCO filters. The retentates were repeatedly washedwith 3 to 5 volumes of water. The retentates for each molecular weightfilter were assayed for GH61 cellulolytic enhancing activity accordingto Example 3 with the following exceptions: 5% (v/v) of each retentatewas added to saccharification reactions of microcrystalline cellulose(AVICEL®), and the extent of hydrolysis was determined at 1 and 6 daysof saccharification. FIG. 6 shows the fractional hydrolysis of AVICEL®by the T. reesei cellulase composition with added T. aurantiacus GH61Apolypeptide as indicated, in the presence of the filter rententatesindicated. FIG. 6 shows that the GH61 polypeptide-enhancing factor wasnot retained, or was only marginally retained by a 3 kDa filter.Increasing concentrations of GH61 polypeptide increased the hydrolysisof AVICEL® in the presence of flow-through from the 3 kDa MWCO filter,whereas a decrease, or no change in hydrolysis with GH61 polypeptideconcentration was observed when incubated in the presence of retentatesfrom molecular weight filters of 3 kDa and greater. These data, combinedwith the data presented in FIG. 5A, indicated that the nominal molecularweight of the compounds facilitating GH61 polypeptide cellulolyticenhancing activity may lie between 1 and 3 kDa, and that molecularweight separation of the NREL acid-pretreated corn stover liquor canseparate enhancing from inhibitory factors.

Example 20 Effect of Thermoascus aurantiacus GH61 Polypeptide andPretreated Corn Stover Liquor Enhances Cellulolytic Activity ofIndividual Cellulases on AVICEL®

Individual monocomponent cellulases Cel5A endoglucanase II, Cel6Acellobiohydrolase, Cel7A cellobiohydrolase, and Cel7B endoglucanase Ifrom Trichoderma reesei and beta-glucosidase from Aspergillus oryzaewere assayed for enhancement of their ability to hydrolyzemicrocrystalline cellulose when incubated with the Thermoascusaurantiacus GH61A polypeptide with or without 5% NREL acid-pretreatedcorn stover liquor. AVICEL® hydrolyses were performed according toExample 3, with the following exceptions. Instead of the Trichodermareesei cellulase composition, purified T. reesei monocomponents andpurified Aspergillus oryzae beta-glucosidase were used at aconcentration of 10 mg of enzyme protein per g cellulose. Alternatively,mixtures of monocomponent cellulases were used, with each cellulasedosed at 10 mg of enzyme protein per g cellulose. Saccharificationreactions were performed with or without 1 mg of the T. aurantiacusGH61A polypeptide per g cellulose and with or without 5% NREL pretreatedcorn stover liquor at 50° C. for 7 days.

FIG. 7A shows the fractional hydrolysis achieved by each monocomponentand monocomponent mixture at 3, 5, and 7 days of hydrolysis, with andwithout the T. aurantiacus GH61A polypeptide in the presence ofacid-pretreated corn stover liquor. In each case, in the absence ofliquor, no GH61 polypeptide-dependent enhancement was observed (data notshown). Conversely, when acid-pretreated corn stover liquor was present,the T. aurantiacus GH61A polypeptide enhanced the cellulase activity ofeach monocomponent or monocomponent mixture by 5% or more at 7 days ofhydrolysis.

FIG. 7B shows the enhancement of cellulase activity for eachmonocomponent arising from the T. aurantiacus GH61A polypeptide in thepresence of NREL acid-pretreated corn stover liquor at 3, 5, and 7 daysof hydrolysis, which is given by the ratio of fractional hydrolysis inthe presence to the absence of the GH61 polypeptide:

$\begin{matrix}{{{GH}\; 61\mspace{14mu}{effect}} = \frac{\%\mspace{14mu}{conversion}_{({{{+ {GH}}\; 61} + {liquor}})}}{\%\mspace{14mu}{conversion}_{({{{no}\mspace{14mu}{GH}\; 61} + {liquor}})}}} & \left( {{Equation}\mspace{14mu} 3} \right)\end{matrix}$Enhancement of hydrolysis by the GH61 polypeptide yields a ratio >1;inhibition of hydrolysis yields a ratio <1, and no effect on hydrolysisyields a ratio=1

In each case at 5 and 7 days of hydrolysis, every monocomponent andevery monocomponent mixture had enhanced cellulolytic activity,indicated by a GH61 effect>1. The mixtures containing 3 to 5 cellulasecomponents had the highest overall cellulase concentration in thereactions, thus had the highest fractional hydrolysis, and the lowestapparent GH61 effect. Despite this, the T. aurantiacus GH61A polypeptidestill provided an enhancement of 1.05±0.00604 for the 5-componentcellulose mixture (Cel5A endoglucanase II, Cel6A cellobiohydrolase,Cel7A cellobiohydrolase, and Cel7B endoglucanase I from T. reesei andbeta-glucosidase from Aspergillus oryzae) at 7 days of hydrolysis. Thelargest relative enhancements by the T. aurantiacus GH61A polypeptide toAVICEL® saccharification by cellulose monocomponents or mixtures thereofwere observed for GH61 polypeptide enhancement of T. reesei Cel7Acellobiohydrolase (1.33±0.127), or mixtures containing the T. reeseiCel7A cellobiohydrolase and T. reesei Cel5A endoglucanase II(1.33±0.00719).

Example 21 Enrichment of NREL Acid-Pretreated Corn Stover LiquorComponents

NREL acid-pretreated corn stover liquor was fractionated by adsorptionto microcrystalline cellulose as described below. NREL acid-pretreatedcorn stover liquor (1.2 liters) was incubated with 10 g of AVICEL®overnight at room temperature. The supernatant fraction was removed byvacuum filtration through Whatman #3 filter paper. The AVICEL® andadsorbed liquor components were washed 6 times with 500 ml ofacetonitrile (occasionally acetone was used), followed by elution with 2liters of water in 300-400 ml fractions. The liquor components that incombination with a GH61 polypeptide demonstrated a GH61polypeptide-dependent enhancement of cellulolysis by the Trichodermareesei cellulase composition were largely eluted in the first 2 waterelution fractions (FIG. 8B), though later elution fractions containedsome small amount of residual liquor. Water-eluted liquor componentswere concentrated approximately 10-fold using a Macrosep 1 kD Omegacentrifuge filter (Pall Corporation, East Hills, N.Y., USA) and appliedto an 8.0 mm×300 mm Shodex Sugar SP0810 HPLC chromatography column(Showa Denka America, Inc., N.Y., USA) using an AGILENT® 1100 HPLC andCHEMSTATION® software (Agilent Technologies, Santa Clara, Calif., USA)and separated by isocratic elution with water at a flow rate of 0.5 mlper minute at 80° C. for 50 minutes, and collecting 250 μl fractions in2.2 ml, 96-deep well plates (Axygen, Union City, Calif., USA). Repeated50 μl injections of the liquor followed by repeated fraction collectionyielded approximately 8 ml of each fraction. Peaks were identified bydiode array detection at 210, 280 and 340 nm using a CHEMSTATION®,AGILENT® 1100 HPLC (Agilent Technologies, Santa Clara, Calif., USA)(FIG. 8, dashed lines).

Peak fractions were assayed according to Example 3 with the followingexceptions. 400 μl of each fraction were added to the hydrolysisreactions containing 4 mg of the T. reesei cellulase composition and 1mg of Thermoascus aurantiacus GH61A polypeptide. Aliquots were removedand analyzed for sugar content at 1, 3, and 10 days of saccharification.Control reactions containing the original NREL acid-pretreated cornstover liquor and control reactions containing no liquor and no GH61polypeptide were run in parallel. FIG. 8B shows that addition of thevarious fractions resulted in fluctuation between fractions around amean fractional hydrolysis value of 0.617 (FIG. 8, solid lines). Severalpeaks of activity consisting of multiple sequential fractions with highcellulolytic activity in the presence of the T. aurantiacus GH61Apolypeptide were observed, including one broad peak of activity centeredat fraction 7C. This peak corresponded with a peak in A₍₂₈₀₎. The extentof the cellulolytic enhancement by the fraction appeared small,indicating that the amount fractionated was very small. Absorbance at280 nm was consistent with contents of the fractions possessing aromaticcharacteristics.

Example 22 Effect of NREL Pretreated Corn Stover Liquor Fractions andthe Thermoascus aurantiacus GH61A Polypeptide on Hydrolysis ofMicrocrystalline Cellulose by the Trichoderma reesei CellulaseComposition

Pooled, lyophilized fractions of the NREL acid-pretreated corn stoverliquor were each added to approximately 25 mg per ml of AVICEL® in 700μl of 50 mM sodium acetate pH 5.0 in the presence of 3 mM calciumchloride with 37.5 μg of the Thermoascus aurantiacus GH61A polypeptideper ml and were incubated in 1.7 ml microcentrifuge tubes at 50° C. withshaking at 1500 rpm for 48 hours in a Thermomixer (Eppendorf, Hamburg,Germany). Following the incubation, the samples were centrifuged at31,000 rpm in a microcentrifuge for 5 minutes. A series of AVICEL®masses were weighed out and suspended in 700 μl of equivalent buffer,incubated for an equivalent length of time, and pelleted equivalently.The height of the AVICEL® was measured using a transparent ruler, andassuming the microcentrifuge tubes were roughly equivalent in volume,the volume of the conical portion of the tube was given by the followingequation:V=⅓_(πr) ² h  (Equation 4)Thus the volume of the AVICEL® scales proportionally with the height ofthe pellet, and a measurement of pellet height could therefore be usedto approximate the volume.

FIG. 9 shows a standard curve of AVICEL® height vs. mass of AVICEL®. Themeasurement of the pellet heights for various masses of AVICEL® scaledlinearly within a volume region spanning the conical portion of thetube.

FIG. 10 shows the height of the AVICEL® for several of the pooled NRELacid-pretreated corn stover liquor HPLC fractions incubated with the T.aurantiacus GH61A polypeptide. One of these pooled fractions (denoted6H-7A) incubated with the T. aurantiacus GH61A polypeptide was notablyhigher, 9 mM compared with an average of all other samples of 5.2 mM, a1.7-fold larger volume. The AVICEL® in this sample had swollen to avolume equivalent to an AVICEL® mass of approximately 90 mg. Pooledfractions 6H-7A along with the T. aurantiacus GH61A polypeptide clearlyinduced swelling of the AVICEL®, whereas inclusion of other liquorfractions with the GH61 polypeptide did not induce swelling.

The supernatants from the AVICEL® incubations were then decanted off andthe pellet dried using a GeneVac EZ-2 Plus® vacuum concentrator (GenevacInc., Gardiner, N.Y., USA). The dried residues were dissolved indeuterated water and were analyzed by ¹H NMR using a VARIAN®MercuryV×400 MHz NMR (Varian, Palo Alto, Calif., USA). While thecomponents of the liquor could not be identified by NMR spectroscopy,the NREL liquor fractions that produced swelling of the AVICEL® onincubation with the T. aurantiacus GH61 polypeptide generated NMR peakscorresponding to saccharide chemical shifts.

The solid residual cellulose was then tested for reducing end contentusing a method modified from Zhang and Lynd, 2005, Biomacromolecules 6:1510-151. The cellulose was washed in 1 ml of 1.1% sodium dodecylsulfate (SDS) and incubated at 95° C. for 10 minutes with shaking, andthen the suspension was pelleted by centrifugation at 31,000 rpm in amicrocentrifuge. The SDS solution was decanted off and the pellet waswashed by repeated resuspension in 1.5 ml of 70% ethanol and pelleting.The pellets were finally incubated with a 1:1 solution of H₂O and BCAworking solution (1:1 mixtures of 0.624 g of CuSO₄.5H₂O, and 0.631 g ofL-serine per 500 ml of H₂O with 0.971 g of disodium2,2′-bichinchoninate, 27.12 g of Na₂CO₃, and 12.1 g of NaHCO₃ per 500 mlof H₂O) at 65° C. for 30 minutes. The concentration of reducing ends wasdetermined by comparison to a standard curve generated by serialdilution of glucose in the same BCA working solution and measurement ofA_((600nm)) in a 96-well microtiter plate using a POWERWAVE X™microplate spectrophotometer (Biotek Instruments, Winooski, Vt. USA).

FIG. 11A shows the glucose reducing end standard curve of A_(600 nm) vs.glucose concentration. The concentration of reducing ends in the T.aurantiacus GH61A polypeptide incubated samples was calculated from thisstandard curve. FIG. 11B shows the number of reducing end equivalentsfor AVICEL® incubated with the GH61 polypeptide and the indicated NRELacid-pretreated corn stover HPLC fractions. The same NRELacid-pretreated corn stover fractions that induced GH61polypeptide-dependent swelling of the AVICEL®, showed a much higherapparent concentration of reducing end equivalents in the insoluble,washed residual cellulose suggesting that hydration of these reducingends was the likely cause of the swelling of the cellulose.

Example 23 Enrichment of Acid-Pretreated Xylan Components

Xylan was acid-pretreated and then fractionated by HPLC chromatographyaccording to Example 21 for NREL acid-pretreated corn stover liquor withthe following exceptions. Acid pretreated xylan was generated byincubating 45 g of beechwood xylan with 400 g of 1.1% H₂SO₄ at 190° C.for 2 minutes in a a 1 gallon, high-pressure horizontal stirred reactor(Parr Instrument Company, Moline, Ill., USA). No preincubation withcellulose and organic phase wash was performed. The acid-pretreatedxylan was filtered with a 3 kDa MWCO VIVASPIN 20® centrifuge filter (GEHealthcare, Piscataway, N.J., USA), retained on a Macrosep 1 kD Omegacentrifuge filter (Pall, Ann Arbor, Mich.), washed with 3-fold volumesof deionized water, and applied to a 8.0 mm×300 mm Shodex Sugar SP0810chromatography column (Showa Denka America, Inc., N.Y., USA) using anAGILENT® 1100 HPLC and CHEMSTATION® software, and separated by isocraticelution with water at a flow rate of 0.5 ml per minute at 80° C. for 50minutes, collecting 250 μl fractions in 2.0 ml, 96-deep well plates(Axygen, Union City, Calif., USA). Repeated 100 μl injections of theliquor followed by repeated fraction collection yielded approximately 5ml of each fraction, which were pooled and lyophilized to dryness. Laterseparations included an overnight incubation of the acid-pretreatedxylan with endoxylanase (SHEARZYME™, Novozymes A/S, Bagsvaerd, Denmark)in 50 mM sodium acetate pH 5.0 at 50° C. to hydrolyze xylo-oligomers,followed by removal of the enzyme with a 3 kDa MWCO VIVASPIN 20®centrifuge filter prior to chromatography. Thermoascus aurantiacus GH61Apolypeptide titrations to AVICEL® hydrolysis reactions containing eitherthe endoxylanase-treated or the untreated xylan liquor were performedaccording to Example 3 and confirmed that endoxylanase treatment had notaltered the effects of acid-pretreated xylan on GH61 polypeptideenhancement of cellulolysis in these samples. Absorbance of the elutedfractions was not determined. Fractions were assayed by dissolution ofthe lyophilized fractions in 200 μl of water, and addition of 100 μl tosaccharification reactions according to Example 3, containing 4 mg ofthe T. reesei cellulase composition and 1 mg of the T. aurantiacus GH61Apolypeptide, or no GH61A.

FIG. 12 shows the hydrolysis of AVICEL® by the T. reesei cellulasecomposition with supplemented T. aurantiacus GH61A polypeptide and theHPLC fractions as indicated. This elution profile demonstrated a broadelution peak of fractions that in the presence of the GH61 polypeptideresulted in higher hydrolysis by the T. reesei cellulase composition.Thus, these fractions contained compounds that in the presence of GH61polypeptide promoted GH61 polypeptide-dependent cellulolyticenhancement. Several inhibitory fractions were also observed, as isevident in FIG. 12, fraction 5D. The elution of the components thatfacilitate GH61 polypeptide cellulolytic enhancement centered on thesame fractions that had previously been shown to facilitate GH61polypeptide cellulolytic enhancement in chromatography of NRELpretreated corn stover liquor (Example 15). It is likely that the widthof the elution was due to overloading of the HPLC column and concomitantloss of theoretical plate. This hypothesis was further supported by themagnitude of the enhancement in cellulolysis by the HPLC fractionsexamined, which was much higher than observed following NRELacid-pretreated corn stover liquor.

The fractions that had higher fractional hydrolysis in the presence ofthe Thermoascus aurantiacus GH61A polypeptide were analyzed by LC-MS inthe following manner. Samples were diluted in 0.1% formic acid to 20 μgper ml approximate concentrations. Samples were then either filteredusing Ultrafree-MC centrifugal filter devices (Millipore Corporation,Billerica, Mass., USA) or were centrifuged for 10 minutes at 21,000×gand then stored at 4° C. if analysis was not performed immediately.UPLC® tandem mass spectrometry, was performed using a Q-T of Micron™hybrid orthogonal quadrupole time-of-flight mass spectrometer (WatersMicromass MS Technologies, Milford, Mass., USA) using MASSLYNX™ softwareversion 4.1 (Waters Micromass MS Technologies, Milford, Mass., USA). TheQ-TOF MICRO™ was fitted with an ACQUITY UPLC® using a 1.0×50 mm, C18,1.7 μm, BEH Acquity column (Waters Corp, Milford, Mass., USA) to permitchromatographic separation of analytes. The following elution gradientwas applied over a 37 minute interval ata flow rate of 100 μl perminute: 0-15 minutes from 1-15% acetonitrile with 0.1% formic acid,15-20 minutes from 15-40% acetonitrile with 0.1% formic acid, 20-25minutes from 40-80% acetonitrile with 0.1% formic acid, 25-30 minuteswith 80% acetonitrile with 0.1% formic acid, 30-31 minutes from 80-1%acetonitrile with 0.1% formic acid, and 31-37 minutes with 1%acetonitrile with 0.1% formic acid. Elution was monitored at 280 nmthrough a diode array detector and eluents from the column wereintroduced directly into the Q-TOF MICRO™ via electrospray ion source. Acone voltage of 20 volts was typically used and the collision energy wasvaried in the range of 5-15 volts. Data were acquired in survey scanmode from a mass range of m/z 50 to 1000 with switching criteria for MSto MS/MS that included an ion intensity of greater than 50.0 counts persecond. The acquired spectra were combined, smoothed, and centered in anautomated fashion. Analytes were identified by comparison to spectra ofstandard compounds when possible. Standard compounds were analyzedbefore and after the samples to confirm the mass accuracy and retentiontime stability of the sample analyses.

FIG. 13 shows a representative LC-MS chromatogram of a T. aurantiacusGH61A polypeptide cellulolytic enhancing acid-pretreated xylan HPLCfraction. It is clear from the number of peaks that significantheterogeneity was observed. The bulk of the components were eluted fromthe LC in the first 6-minutes, suggesting a highly polar or chargedcomposition.

Example 24 Using GH61 Polypeptide-Binding Affinity to Enrich NRELAcid-Pretreated Corn Stover Liquor for Components that are Functional inGH61 Polypeptide-Dependent Cellulolytic Enhancement

Thermoascus aurantiacus GH61A polypeptide affinity was used as a meansto enrich NREL acid-pretreated corn stover liquor components that bindto the GH61 polypeptide. Seventy mg of the T. aurantiacus GH61Apolypeptide were incubated overnight with 50 ml of liquor. The GH61polypeptide-bound liquor components were separated from the freecomponents by ultracentrifugation using a 10 kDa MWCO VIVASPIN 20®centrifuge filter, concentrating the protein and protein-bound fraction10-fold, followed by washing with an equivalent volume of water to thestarting material (50 ml total volume), and repeating 5-times. Finally,the protein was denatured by incubation at 90° C. for 30 minutes, andcentrifuged using a 10 kDa MWCO VIVASPIN 20® centrifuge filter. Theflow-through from the filter was analyzed using liquid chromatographymass spectrometry according to Example 23, with electrospray massspectrometry performed in both positive and negative ionization modes.

FIG. 14 shows the LC-MS chromatogram of the NREL acid-pretreated cornstover liquor components that bound to the T. aurantiacus GH61Apolypeptide and were eluted by denaturation. Both positive and negativemode ion chromatographs were combined, yielding a small set of uniquemass ions, many of which could be excluded as dimers, fragments oflarger parent ions or contaminants derived from the buffer or from thecentrifuge columns, leading to identification of approximately 30 uniquemass ions of reasonable intensity. Based on the monoisotopic massesobtained, chemical formulae were determined and a database search ofchemical compounds (ChemSpider, Royal Society of Chemistry) yielded alist of putative compounds for GH61 polypeptide assay. In thechromatograms, 3 broadly classified sets of compounds could beidentified: the first set eluted in the first 2-minutes, had smallermass and were likely small polar or charged compounds; a second lowerabundance set, corresponding to a broadly varied set of molecularweights but likely to be less polar, eluted with intermediate retentiontimes; and finally a series of larger molecular weight ions elutedbetween 12 and 20 minutes, corresponding to less polar or highermolecular weight compounds. It was observed that the search ofmonoisotopic masses frequently yielded compounds consistent with plantflavonols, flavanols, oxidized flavonoids, oxidized flavanols andsimilar compounds, thus a large set of these compounds were tested forGH61 cellulolytic enhancing activity.

Example 25 Effect of NREL Pretreated Corn Stover Liquor and theThermoascus aurantiacus GH61A Polypeptide on Generating SolubleCellodextrin From Microcrystalline Cellulose or Phosphoric Acid-SwollenCellulose

Monosaccharide, disaccharide, and aldonic and uronic acid standards(Sigma-Aldrich, St. Louis, Mo., USA) were dissolved in water at 1 mg perml and were diluted to 50 μg per ml in 10 mM NaOH. Cellooligosaccharides(Sigma-Aldrich, St, Louis, Mo., USA) were dissolved in water at 25 to 30mg per ml and were diluted as above. Xylooligosaccharides (MegazymeBray, Co. Wicklow, Ireland) were dissolved in water at 10 to 30 mg perml and were diluted as above. Lactobionic acid was purchased fromSigma-Aldrich (St. Louis, Mo., USA), dissolved in water at 2.5 mg perml. The aldonic acids of each cellooligosaccharide were generated byincubation of 17 μg of Humicola insolens cellobiose dehydrogenase (WO2010/080527) per ml with 2.5 mg per ml of each cellooligosaccharide in50 mM sodium acetate pH 5.0 at 50° C. and 10 mM dichloroindophenol(DCIP; Sigma-Aldrich, St. Louis, Mo., USA) was added incrementally. Theequivalence point was achieved when DCIP was not reduced by thesolution, and the color remained purple. Formation of the aldonateproducts was confirmed by LC-MS according to Example 25. Cellobiosedehydrogenase was then removed by ultrafiltration through a VIVASPIN 20®10 kDa MWCO centrifuge filter.

The Thermoascus aurantiacus GH61A polypeptide was incubated at 37.5μg/ml with either 29.5 mg per ml microcrystalline cellulose (AVICEL®™,Sigma Aldrich, St. Louis, Mo., USA) or 1.2% (w/w) phosphoricacid-swollen cellulose (PASC) in 50 mM sodium acetate, 1 mM MnSO₄ for 5days at 50° C. with or without 10% (v/v) NREL pretreated corn stoverliquor. NREL pretreated corn stover liquor was extracted according toExample 2. Phosphoric acid swollen cellulose was prepared from AVICEL®PH101 using the protocol described by Zhang et al., 2006,Biomacromolecules 7: 644-648. Control incubations containing AVICEL®alone, PASC alone, AVICEL® or PASC with NREL acid-pretreated corn stoverliquor in the absence of the T. aurantiacus GH61A polypeptide wereperformed in the same manner. The samples were then filtered through a0.22 μm centrifuge filter, and the filtrates were diluted 1:10 in 1 mlfinal volume of 10 mM NaOH and analyzed by DIONEX® Ion Chromatographywith pulsed amperometry detection (IC-PAD, Dionex Corporation,Sunnyvale, Calif.) using DIONEX® Chromeleon or PeakNet Software. Ten μlof 2.5 mg per ml lactobionic acid was added as an external loadingcontrol. Chromatographic separation was obtained using a PA-20 column(Dionex Corporation, Sunnyvale, Calif., USA), with relevant guard,borate and amine-trap pre-columns, and elution was achieved with anisocratic gradient of 13 mM sodium hydroxide, 2.5 mM sodium acetate for20 minutes, followed by a linear gradient from 13 to 50 mM NaOH,isocratic gradient for 10 minutes and linear gradient from 0.5 to 40 mMsodium acetate in 50 mM NaOH.

FIG. 15A shows the chromatograms of AVICEL® and PASC incubations withGH61 polypeptide, with GH61 polypeptide and pretreated corn stoverliquor, and control incubations. The chromatograms indicated thatsoluble oligosaccharide products were evolved from the incubationscontaining the GH61 polypeptide, liquor, buffer, and cellulose in bothPASO and microcrystalline forms. The arrows indicate unique elutionpeaks that were present only in the incubations containing the full setof components. FIG. 15B shows a comparison of chromatogram of AVICEL®incubated with the T. aurantiacus GH61A polypeptide, acid-pretreatedcorn stover liquor, and buffer to the chromatograms of standardcompounds. FIG. 15B indicated that the retention times of the novelproducts were consistent with those of cellopentaose, cellotetraose, andcellotriose. Three additional elution peaks were also evident; a largepeak with retention time of 49 minutes matched no available standard butwas likely cellohexaose based on its retention time in comparison to thecellooligosaccharides and aldonic acid standards. The relative peakintensities indicated that the major product may be cellotetraose,though the sparing solubility of cello-oligosaccharides of DP≥6 may haveunderstated the actual production of these oligomers.

Example 26 Effect of Water-Extracted and Alkaline Pretreated Corn StoverLiquors in Combination With the Thermoascus aurantiacus GH61A onCellulolysis by the T. reesei Cellulase Composition

The Thermoascus aurantiacus GH61A polypeptide was assayed forcellulolytic enhancing activity according to Example 3 with 10% (v/v)added liquor from either alkaline pretreated corn stover or 10% (v/v)added liquor from water-extracted acid-pretreated corn stover accordingto Example 2. Alkaline pretreated corn stover was prepared in thefollowing manner. Milled and washed, raw PCS was treated with 4%, 6%, 8%or 10% (w/w) NaOH for 1 hr at 90° C. in a 2-L stirred tank reactor (IKAWorks, Inc., Wilmington, N.C., USA). Water extracted corn stover liquorswere generated in the following manner. Raw corn stover was milled in aWILEY® Mill (Model 4; Thomas Scientific, Swedesboro, N.J., USA) with anominal screen size of 2 mm. The milled stover was sieved through a #40mesh screen and the fines were discarded. A Dionex Accelerated SolventExtractor (ASE) 350 (Dionex Corporation, Sunnyvale, Calif., USA)instrument was used for all pretreatments with two cycles for eachpretreatment. The first cycle of each pretreatment was a pressurized hotwater extraction run in standard flow mode of operation. The secondcycle of the pretreatment was the same for each experimental sample, andwas a mid-severity dilute acid pretreatment run in pressure solventsaver mode of operation. Approximately 20.0 g of the sieved corn stoverwere packed into a 100 ml stainless steel extraction cell. During thepressurized hot water extraction the stover was extracted attemperatures of 100-170° C. in 10° C. increments for a static reactiontime of 7 minutes. After extraction the cell was purged with nitrogenfor 60 seconds, and all extraction liquor collected. Static pretreatmenttime was defined from the time the internal temperature of the cellreached equilibrium with the heating chamber. Liquors from the first,water-extraction step were filtered using a 10 kDa MWCO VIVASPIN 20®centrifuge filter and were pH adjusted to 5.0 by addition of NaOH orHCl, prior to use.

As shown previously, GH61 polypeptide titrations in the absence of addedliquors did not significantly enhance microcrystalline cellulosehydrolysis by the T. reesei cellulase composition (Examples 15, 16, and18). FIG. 16A shows the fractional hydrolysis of AVICEL® by the T.reesei cellulase composition with increasing concentrations of the T.aurantiacus GH61A polypeptide in the presence of liquors from variousseverities of alkaline pretreated corn stover. Addition of alkaline cornstover liquors increasing in severity from 4% NaOH to 10% NaOH showedreduced fractional hydrolysis, thus increasing inhibition in the absenceof the GH61 polypeptide. Unlike acid-pretreated corn stover liquors,however, addition of the GH61 polypeptide in the presence of alkalineliquors provided no substantial improvement over saccharificationswithout liquor.

The higher severity alkaline pretreated corn stover liquors incombination with the T. aurantiacus GH61A polypeptide did provide asmall amount of cellulolytic enhancing activity at 7 days of hydrolysis.Comparing zero to 50% GH61 polypeptide additions, hydrolysis in thepresence of 6% NaOH liquor was 0.473±0.00483 and 0.488±0.00724, 8% NaOHwas 0.454±0.00352 and 0.485±0.000119, and 10% NaOH was 0.445±0.00625 and0.476±0.00732 The addition of the T. aurantiacus GH61A polypeptide wassufficient to partially mitigate the inhibition arising from the liquoradditions; as the hydrolysis in the absence of liquor was 0.502±0.0109and 0.499±0.00642 at zero and 50% (w/w) GH61A polypeptide, respectively.

FIG. 16B shows the fractional hydrolysis of AVICEL® by the T. reeseicellulase composition with increasing concentrations of the T.aurantiacus GH61A polypeptide in the presence of corn stover liquorsextracted with water at the indicated temperatures. Pressurizedwater-extracted corn stover liquors did not appreciably enhancefractional hydrolysis in the presence of the GH61 polypeptide.Water-extracted liquors generated at 150° C. and above show someinhibition, both in the absence and presence of the GH61 polypeptide.These data indicated that pressurized water-extraction did not extractthe liquor components required to observe GH61 polypeptide cellulolyticenhancing activity on microcrystalline cellulose.

Example 27 Effect of Acid-Pretreated Xylan, Acid-Pretreated BiomassComponent Mixtures Containing Xylan, or Acid-Pretreated MonosaccharideComponents of Xylan Including Xylose and Arabinose in Combination WithThermascus aurantiacus GH61A Polypeptide on Cellulose Hydrolysis by theTrichoderma reesei Cellulase Composition

The cellulolytic enhancing effect of the T. aurantiacus GH61Apolypeptide on microcrystalline cellulose was assayed with components ofbiomass that had been acid-pretreated.

The Thermoascus aurantiacus GH61A polypeptide was assayed forcellulolytic enhancing activity according to the procedure described inExample 3 with the following exceptions: hydrolysis reactions wereperformed with 10% (v/v) added liquor from acid-pretreated corn stover(Example 2) or with 10% (v/v) added liquor from acid-pretreated biomasscomponents including xylan, cellulose, protein, lipid, monosaccharide,and combinations thereof. AVICEL® (1.8 g), oatspelt xylan (1.1 g; TCI),lignin (0.9 g; MeadWestvaco), corn gluten (0.25 g; Sigma Aldrich, St.Louis, Mo., USA), gallic acid (0.25 g; Sigma Aldrich, St. Louis, Mo.,USA), ferulic acid (0.25 g; Sigma Aldrich, St. Louis, Mo., USA), or cornoil (0.25 g; Sigma Aldrich, St. Louis, Mo., USA) were pretreated bydissolution in 25 ml of 1% H₂SO₄ and incubation in an SBL-2 fluidizedsand bath reactor with TC-8D Temperature Controller (Techne Inc.,Burlington, N.J., USA) for a 5 minutes temperature ramp followed by 5minutes at 190° C. Aliquots from saccharification reaction were removedfor analysis at 1 and 5 days. Liquors were filtered using a 10 kDa MWCOVIVASPIN 20® centrifuge filter and pH adjusted to 5.0 by addition ofNaOH or HCl, prior to use.

FIGS. 17A and 17B show fractional hydrolysis of AVICEL® by the T. reeseicellulase composition with various concentrations of the T. aurantiacusGH61A polypeptide in the presence of liquors derived fromacid-pretreatment of various biomass components. Titration of the T.aurantiacus GH61A polypeptide from 0 to 50% (w/w) addition to the T.reesei cellulase composition yielded, in the absence of liquor, noenhancement of AVICEL® hydrolysis (Example 15, 16, 18, and FIG. 19B).Increasing concentrations of the T. aurantiacus GH61A polypeptideincreases fractional hydrolysis in the presence of a subset of theliquors examined. Small GH61 polypeptide concentration-dependenthydrolysis enhancements were observed in the presence of liquors ofacid-pretreated lignin, lipid, protein, cellulose, lignin modelcompounds such as gallic acid, and hemicellulase components such asferulic acid. The extent of GH61 polypeptide enhancement of fractionalhydrolysis in the presence of acid-liquors was from 0.390±0.007 to0.435±0.010 in the presence of lignin liquor, from 0.305±0.013 to0.341±0.016 in the presence of corn oil liquor, 0.493±0.007 to0.524±0.004 in the presence of corn gluten liquor, 0.134±0.021 to0.184±0.023 in the presence of gallic acid liquor, 0.352±0.011 to0.397±0.029 in the presence of ferulic acid liquor at 7 days ofhydrolysis. Conversely, liquor derived from acid-pretreatment of xylanyielded a substantial GH61 polypeptide concentration-dependentenhancement of cellulolysis, from 0.390±0.009 to 0.540±0.013 at 7 daysof hydrolysis. Where possible, biomass components had been acidpretreated at their respective concentrations in NREL acid-pretreatedcorn stover, thus the observed GH61 cellulolytic enhancements shouldscale proportionally with their contributions to enhancements inpretreated corn stover. The bulk of the apparent GH61 polypeptideconcentration-dependent cellulolytic enhancements on cellulose are thusderived from the xylan components.

Example 28 Effect of Residual Liquors Post-Fermentation in CombinationWith the Thermoascus aurantiacus GH61A Polypeptide on CelluloseHydrolysis by the T. reesei Cellulase Composition

The Thermoascus aurantiacus GH61A polypeptide was assayed forcellulolytic-enhancing activity according to Example 3 with thefollowing exceptions: 10% (v/v) added liquor from post-fermentationresidues was added, and aliquots were removed at 1, 3, and 7 days ofsaccharification. Liquors were filtered using a 10 kDa MWCO VIVASPIN 20®centrifuge filter and pH adjusted to 5.0 by addition of NaOH or HCl,prior to use. Fermentation liquors were obtained as follows: 20% totalsolids NREL unwashed, unmilled pretreated corn stover was hydrolyzed by5 mg per gram cellulose of a T. reesei cellulase composition for 5-days.The hydrolysate was fermented for 2 days using a Saccharomycescerevisiae strain comprising a xylose isomerase gene (WO 2003/062430) at3% cell density for 2 days. Fifty ml of the fermentation liquor wasfiltered as described and assayed for GH61 polypeptideconcentration-dependent cellulolytic enhancement activity.

FIG. 18 shows the fractional hydrolysis of AVICEL® by the T. reeseicellulase composition with various concentrations of the T. aurantiacusGH61A polypeptide in the presence of post-fermentation liquors. Theaddition of increasing T. aurantiacus GH61A polypeptide concentrationssignificantly enhanced hydrolysis of AVICEL® in the presence of thepost-fermentation liquors from 0.246±0.007 to 0.265±0.007 at 1 day ofhydrolysis and from 0.616±0.006 to 0.895±0.002 at 7 days of hydrolysis.

Example 29 Effect of Corn Stover Liquors From Increasing Severity ofAcid Pretreatments in Combination With the Thermoascus aurantiacus GH61APolypeptide on Cellulose Hydrolysis by the T. reesei CellulaseComposition

The Thermoascus aurantiacus GH61A polypeptide was assayed forcellulolytic enhancing activity according to Example 3 with 10% (v/v)added liquor from acid-pretreated corn stover or microcrystallinecellulose (AVICEL®) of various severity pretreatments. Liquors werefiltered using a 10 kDa MWCO VIVASPIN 20® centrifuge filter and pHadjusted to 5.0 by addition of NaOH or HCl, prior to use. Liquors weregenerated using an SBL-2 fluidized sand bath reactor with TC-8DTemperature Controller, incubating corn stover with 1% H₂SO₄ in 25 mland incubating at temperatures between 140° C. and 170° C. for 1 to 5minutes, or by incubating AVICEL® with 1% H₂SO₄ in 25 ml at temperaturesbetween 110° C. and 190° C. for 5 minutes.

FIGS. 19A and 19B show the fractional hydrolysis of AVICEL® containingvarious severity acid-treatments of corn stover by the T. reeseicellulase composition and various concentrations of the T. aurantiacusGH61A polypeptide. Liquors were generated by systematically varying bothtemperature between 140° C. and 170° C. and varying time at eachtemperature between 1 and 5 minutes. For saccharification reactionscontaining liquors produced by acid-pretreatment at temperatures greaterthan 140° C. and 5 minutes, increasing T. aurantiacus GH61A polypeptideconcentrations increased fractional hydrolysis. The higher severitypretreatments, particularly those of 160° C. and above, showed a greaterincrease in fractional hydrolysis from GH61 polypeptide addition thandid the lower severity pretreatments of 150° C. and less. Additionally,higher severity pretreatment liquors were increasingly inhibitory tocellulolysis, thus the greatest overall cellulose conversion wasobtained by addition of high GH61 polypeptide concentrations (50% w/w)at intermediate pretreatment severity, specifically 150° C. for 5minutes, 160° C. for 3-5 minutes, or 170° C. for 1 minute. These datademonstrated that pretreatment of a biomass can be tailored to maximizehydrolysis for a given GH61 polypeptide concentration in a cellulasecomposition. There is an optimum pretreatment condition that balancesGH61 polypeptide cellulolytic-enhancing activity with production ofsoluble inhibitor compounds.

FIG. 19C shows the fractional hydrolysis of AVICEL® containing variousseverity acid-treatments of AVICEL® by the T. reesei cellulasecomposition and various concentrations of the T. aurantiacus GH61Apolypeptide. In calculating the fractional hydrolysis, sugars derivedfrom liquor addition were not subtracted, and contributed up to 3% ofconversion. From FIG. 19C it is clear that liquors derived fromacid-treatment of microcrystalline cellulose in combination with the T.aurantiacus GH61A polypeptide enhanced hydrolysis only marginally.

Example 30 Effect of Xylan Liquors From Increasing Severity of AcidPretreatments in Combination With the Thermoascus aurantiacus GH61APolypeptide on Cellulose Hydrolysis by the T. reesei CellulaseComposition

The Thermoascus aurantiacus GH61A polypeptide was assayed forcellulolytic enhancing activity according to Example 3 with 10% (v/v)added liquor from xylan pretreated with varying severity. Liquors weregenerated by incubation of 1.1 g of either oatspelt xylan or wheat flourwith 1% H₂SO₄ or 3% HCl in 25 ml total volume in an SBL-2 fluidized sandbath reactor with TC-8D Temperature Controller for 10 minutes total; a 5minute ramp period followed by a 5 minute incubation at the indicatedtemperature. Incubation temperatures were varied between 60° C. and 180°C. Pretreatment liquors were filtered using a 10 kDa MWCO VIVASPIN 20®centrifuge filter and pH adjusted to 5.0 by addition of NaOH or HCl,prior to use.

FIGS. 20A and 20B show the effect of acid-pretreated xylan liquors addedto the T. reesei cellulase compositions with increasing concentrationsof the T. aurantiacus GH61A polypeptide. In the absence of the GH61polypeptide, liquors of increasing pretreatment severity wereincreasingly inhibitory to the cellulolysis of AVICEL®, particularlythose generated with 3% HCl. Titration of increasing GH61 polypeptideconcentration in the presence of xylan acid-pretreated at temperaturesgreater than 140° C. led to increasing cellulolysis, and the net changein hydrolysis with GH61 polypeptide addition was higher for higherseverity pretreatments. Hydrolysis was not improved by the GH61polypeptide on addition of xylan pretreated at 60° C. to 120° C.Hydrolysis was improved by 0.057, from 0.349±0.00510 to 0.406±0.00394with 160° C. liquor, by 0.089 from 0.287±0.00233 to 0.376±0.00118 with180° C. liquor, and by 0.0740, from 0.216±0.00225 to 0.293±0.00843 with180° C. HCl derived liquor. The effect of increasing pretreatmentseverity on T. aurantiacus GH61A polypeptide cellulolytic enhancingactivity with xylan liquors was similar to those of corn stoverpretreatment severity (Example 26), and again suggested a maximaloverall hydrolysis was obtained by high GH61 polypeptide concentrationsin the presence of liquors of intermediate severity. In this case, 50%(w/w) T. aurantiacus GH61A polypeptide in the presence of 160° C.acid-pretreated xylan produced the highest cellulose conversion.Additionally, as previously observed for corn stover liquors, thereappeared to be an optimum balance between GH61 polypeptide cellulolyticenhancing activity and the production of soluble inhibitors. Theaddition of a specific concentration and tailoring of pretreatmentseverity can be accomplished for a given GH61 polypeptide concentrationwithin a cellulase composition.

FIG. 20C shows the fractional hydrolysis of AVICEL® containing variousseverity acid-treatments of beechwood xylan by the T. reesei cellulasecomposition and various concentrations of the T. aurantiacus GH61Apolypeptide. From FIG. 20C, it was clear that the maximum conversion wasachieved in the presence of a high concentration of the T. aurantiacusGH61A polypeptide (50%) and liquor produced at 170° C. In the presenceof liquor generated at 190° C., the observed hydrolysis was lower forall concentrations of the T. aurantiacus GH61A polypeptide tested, andin the presence of liquors generated below 170° C.; the conversionlevels reached were not as great. These data suggest that treatmentseverity of some substrates may be optimized to generate liquors for agiven GH61 polypeptide concentration within a cellulase composition.

Example 31 Effect of Solid-Phase Extracted Acid Pretreated Corn StoverLiquors in Combination With the Thermoascus aurantiacus GH61APolypeptide on Cellulose Hydrolysis by the T. reesei CellulaseComposition

The Thermoascus aurantiacus GH61A polypeptide was assayed forcellulolytic enhancing activity according to Example 3 with thefollowing exceptions. T. aurantiacus GH61 polypeptide was either notadded or was added at 50% (w/w) of total protein and 10% (v/v) solidphase extraction resin-eluted liquor was added to each hydrolysisreaction. Both NREL acid-pretreated corn stover liquor preparedaccording to Example 2 and acid-pretreated xylan prepared according toExample 23, and then pH adjusted to 5.0, were applied to one of twosolid phase extraction cartridges, either a BOND ELUT™ C-18 column(Varian, Palo Alto, Calif., USA) or a STRATA™-X Polymeric column(Phenomenex, Torrance, Calif., USA) equilibrated in water. The sampleswere washed 3-times with 1 ml of water and then eluted with two aliquotsof 600 μl methanol. The eluted samples were diluted back to theiroriginal volumes with water and assayed for GH61 polypeptidecellulolytic enhancing activity.

FIG. 21 shows the fractional hydrolysis of AVICEL® by the T. reeseicellulase composition with and without 50% (w/w) T. aurantiacus GH61Apolypeptide addition in the presence of solid-phase extracted liquors asindicated. The fractional hydrolysis was equivalent within error for allsamples, indicating that no enhancement of cellulolysis from GH61polypeptide addition was observed in the presence of solid-phaseextracted liquors from acid-pretreated corn stover or fromacid-pretreated xylan. A cellulolytic enhancement of the T. reeseicellulase composition was previously demonstrated in the presence ofthese liquors in combination with addition of the GH61 polypeptide,prior to solid-phase extraction. It was therefore concluded that thecompounds present in these liquors that were correlated with theobserved GH61 polypeptide cellulolytic enhancing activity were elutedduring the wash steps.

Example 32 Effect of Electrodialyzed Acid-Pretreated Corn Stover Liquorson Thermoascus aurantiacus GH61A Polypeptide Enhancing the Trichodermareesei Cellulase Composition

The Thermoascus aurantiacus GH61A polypeptide was assayed forcellulolytic-enhancing activity according to Example 3 with thefollowing exceptions: 10% (v/v) NREL pretreated corn stover liquor,acid-pretreated xylan (Example 23) or electrodialyzed NREL pretreatedcorn stover liquor was added to each hydrolysis reaction.Electrodialyzed NREL pretreated corn stover liquor was generated in thefollowing manner. Two kg of NREL pretreated corn stover liquor wasfiltered and the pH was adjusted to 5.0 using concentrated NaOH. Thepretreatment liquor was subjected to concentrating electrodialysis usinga EUR2B pilot scale electrodialysis unit (Ameridia, Somerset, N.J., USA)equipped with a EUR2B-10 stack Ameridia, Somerset, N.J., USA). Thesolution of pH adjusted liquor had a conductance of 16.5 mS/cm and a pHof 5.0 and was charged to the diluate tank. A solution of potassiumnitrate (20 mS/cm, 2.0 kg) was charged to the electrode rinse tank. Adilute solution of NREL pretreatment liquor (0.657 mS/cm) was charged tothe concentrate tank. The solutions described above were circulatedthrough the EUR2B-10 stack at a flow rate of approximately 0.9 gallonper minute (gpm) using a DC power supply set to 14 volts. After 55minutes, the conductivity change in the diluate and concentrate tanksremained stable and the run was ended. The final conductance in theconcentrate tank was 14.01 mS/cm.

FIG. 22 shows that increasing the T. aurantiacus GH61A polypeptideconcentration in the presence of NREL pretreated corn stover liquor,electrodialyzed NREL acid-pretreated corn stover liquor, andacid-pretreated xylan increased the fractional hydrolysis of AVICEL®. Atthe highest concentrations of GH61 polypeptide, the electrodialyzed NRELliquor showed slightly lower conversion than the undialyzed sample,0.541±0.0006 compared with 0.524±0.0118, though at intermediate GH61polypeptide concentrations the conversion was higher for theelectrodialyzed liquor, 0.467±0.00671 compared with 0.502±0.00215.

Example 33 Effect of Addition of Acid-Pretreated Corn Stover Liquors andThermoascus aurantiacus GH61A Polypeptide to Non-Acid Pretreated BiomassFeedstocks

The Thermoascus aurantiacus GH61A polypeptide was assayed forcellulolytic-enhancing activity according to Example 3 with thefollowing exceptions: 5% (v/v) added NREL acid-pretreated corn stoverliquor was added to either 3% or 5% total solids of various milledbiomass feedstocks, and aliquots were removed for analysis at 1, 3, and7 days of saccharification. Control incubations of each biomasscontaining either no T. reesei cellulase composition and no GH61polypeptide, or containing GH61 polypeptide in the absence of the T.reesei cellulase composition with and without liquor, and controlincubations containing an equivalent concentration of liquor with the T.reesei cellulase composition and GH61 polypeptide without biomass wereperformed in parallel. The composition of total accessible cellulose ineach biomass was determined by measurement of glucose equivalentsproduced by saccharification by 50 mg of Cellic CTec™ (availableNovozymes A/S, Bagsvaerd, Denmark) over 7 days of hydrolysis. Liquorswere filtered using a 10 kDa MWCO VIVASPIN 20® centrifuge filter and pHadjusted to 5.0 by addition of NaOH or HCl, prior to use. Biomassfeedstocks included raw sugarcane bagasse and corn stovers pretreated inthe following manners: organosolv low severity ethanol, organosolvmedium severity ethanol, organosolv high severity ethanol, andorganosolv glycerol.

FIGS. 23A, 23B, 23C and 23D show the fractional hydrolysis of variousbiomass feedstocks by the T. reesei cellulase composition with varyingT. aurantiacus GH61A polypeptide concentrations, with and withoutsupplemented NREL acid-pretreated corn stover liquor, as indicated. Foreach biomass, addition of 5% (v/v) NREL acid-pretreated corn stoverliquor in the absence of the T. reesei cellulase composition added abackground sugar concentration equivalent to approximately 0.05 increasein apparent hydrolysis, depending on the relative amount of cellulosepresent in each substrate. This was not substantially changed byaddition of the T. reesei cellulase composition or the GH61 polypeptide.Since these liquor-derived sugars are not subtracted from the fractionalhydrolysis values, for each biomass the presence of liquor shifts theapparent hydrolysis to a higher value.

For each biomass, a higher fractional hydrolysis was obtained byaddition of higher T. aurantiacus GH61A polypeptide concentrations from0 to 50% (w/w) total protein to the T. reesei cellulase composition. Forsome of these biomasses, the presence of the NREL acid-pretreated cornstover liquor increased the total cellulolytic enhancement by the T.aurantiacus GH61A polypeptide. This was particularly apparent at 3 daysof hydrolysis (gray bars, FIG. 24), as exemplified by the enhancement oflow and medium severity ethanol organosolv pretreatments. In the absenceof GH61 polypeptide, fractional hydrolysis of low severity ethanolorganosolv corn stover was 0.148±0.00447 without liquor and0.165±0.00126 with liquor, whereas at 50% GH61, fractional hydrolysiswas increased from 0.237±0.00737 to 0.273±0.00113. Similarly, in theabsence of GH61 polypeptide, fractional hydrolysis of medium severityethanol corn stover was 0.179±0.000737 without liquor and 0.190±0.00401with liquor, whereas at 50% GH61 polypeptide, fractional hydrolysis wasincreased from 0.281±0.0136 to 0.328±0.00543. Substrates that showedthis augmentation of the GH61 polypeptide cellulolytic enhancing effectincluded organosolv low and medium severity ethanol and sugarcanebagasse. It appeared that cellulolysis of lower severity pretreatmentbiomass feedstocks were better enhanced by the T. aurantiacus GH61Apolypeptide in the presence of supplemented acid-pretreated biomassliquor. The augmentation of the GH61 polypeptide cellulolytic enhancingactivity on the low severity pretreatment by supplemented liquorappeared most dramatic at the early stages of saccharification.

Example 34 Effect of Addition of Acid-Pretreated Monosaccharides onThermoascus aurantiacus GH61A Enhancement of Trichoderma reeseiCellulase Composition Cellulolysis of Microcrystalline Cellulose

The Thermoascus aurantiacus GH61A polypeptide was assayed forcellulolytic enhancing activity as described in Example 3 with thefollowing exceptions. Saccharification reactions were performed with 5mM MnSO₄, and either 5% or 10% (v/v) liquor derived fromacid-pretreatment of monosaccharides was added. Liquors were generatedin the following manner: 20 mg per ml of each monosaccharide wasincubated with 1% H₂SO₄ in an SBL-2 fluidized sand bath reactor withTC-8D Temperature Controller at 190° C. for 5 minutes, and the resultingliquors were then filtered using a 10 kDa MWCO VIVASPIN 20® centrifugefilter and pH adjusted to 5.0 by addition of NaOH or HCl prior to use.Ozonolysis was performed in the following manner: NREL acid-pretreatedcorn stover was slurried at 15% total solids, and the liquor extractedby vacuum filtration using a glass fiber filter. This liquor wasincubated with a low concentration of ozone for 30 minutes prior to pHadjustment.

FIG. 24 shows the fractional hydrolysis of AVICEL® by the T. reeseicellulase composition with either no GH61A polypeptide or 24% (w/w) T.aurantiacus GH61A polypeptide in the presence of 5% or 10% (v/v) ofvarious acid-pretreated monosaccharides, and with ozone-treated NRELpretreated corn stover liquor. FIG. 25 demonstrates that acid-pretreatedmonosaccharides were strongly inhibitory to cellulolysis by the T.reesei cellulase composition in the absence of GH61 polypeptide. In thepresence of acid-pretreated hemicellulose-derived sugars, e.g., C5sugars, including arabinose and xylose, the fractional hydrolysis wasincreased by addition of the T. aurantiacus GH61A polypeptide. In thepresence of 5% acid-pretreated arabinose, addition of the GH61polypeptide increased fractional hydrolysis from 0.183±0.00372 to0.223±0.00548 at 3 days of hydrolysis, and from 0.229±0.00379 to0.328±0.00272 at 7 days of hydrolysis. Similarly, in the presence of 5%acid-pretreated xylose, addition of the GH61 polypeptide increasedfractional hydrolysis from 0.113±0.0148 to 0.140±0.00356 at 3 days ofhydrolysis, and from 0.117±0.00252 to 0.177±0.00602 at 7 days ofhydrolysis. Conversely, addition of the T. aurantiacus GH61A polypeptidein the presence of acid-pretreated glucose increased fractionalhydrolysis to a much smaller extent, even at the higher concentration of10% (v/v), from 0.172±0.00658 to 0.206±0.00108. Ozone treatment of NRELacid-pretreated corn stover liquor altered the liquor components in sucha manner as to disrupt the ability of the liquor in combination with theGH61 polypeptide to enhance cellulolysis of AVICEL® by the T. reeseicellulase composition. In the presence of liquor, the GH61 polypeptideincreased fractional hydrolysis from 0.522±0.00612 to 0.679±0.00807,whereas in the presence of the ozone-treated liquor, the fractionalhydrolysis was increased only from 0.512±0.00347 to 0.540±0.00956.

Example 35 Enhancement of AVICEL® Cellulolysis by T. reesei CellulasesUsing NREL PCS Liquor and Various GH61 Polypeptides

Microcrystalline cellulose saccharification reactions were performed asdescribed (Example 3), using 29.5 mg of microcrystalline cellulose(AVICEL®) per ml and 4 mg of the T. reesei cellulase composition per gcellulose in 50 mM sodium acetate, 1 mM manganese sulfate at pH 5.0 inthe presence of 10% (v/v) NREL PCS liquor prepared as described (Example2). GH61 polypeptides including Thermoascus aurantiacus GH61Apolypeptide, Aspergillus fumigatus GH61B polypeptide and Penicilliumpinophilum GH61 polypeptide were added between 0 and 2 mg per gcellulose (0 and 50% (w/w) of the T. reesei cellulase compositionconcentration). Alternatively, liquor was added at 10% (v/v) tosaccharifications containing either no GH61 polypeptide or 2 mg of thevarious GH61 polypeptides per g cellulose.

FIG. 25 shows the fractional hydrolysis of AVICEL® by the T. reeseicellulase composition with various concentrations of the indicated GH61polypeptides with 10% (v/v) NREL PCS liquor. Fractional hydrolysis isshown for the T. reesei cellulase composition with Thermoascusaurantiacus GH61A (circles), Aspergillus fumigatus GH61B polypeptide(diamonds) and Penicillium pinophilum GH61 polypeptide (squares) at 1day (open symbols) and 3 days of hydrolysis (closed symbols). Additionof liquor in combination with all the GH61 polypeptides showed anapparent increase in hydrolysis of the cellulose. Thermoascusaurantiacus GH61A polypeptide produced the greatest enhancement ofcellulolysis in the presence of liquor, 0.098±at 7 days of hydrolysis.Conversely, addition of the highest concentration of each of the GH61polypeptides did not enhance cellulolysis in the absence of supplementedliquor. Multiple GH61 polypeptides are thus shown to enhancecellulolysis by T. reesei cellulases in the presence of biomass liquor.

Example 36 Effect of Addition of Kraft (Indulin) Lignin or OxidizedKraft (Indulin) Lignin on the Thermoascus aurantiacus GH61A Enhancementof Trichoderma reesei Cellulase Composition Cellulolysis ofMicrocrystalline Cellulose

Microcrystalline cellulose saccharification were performed as described(Example 3), using 25 mg of microcrystalline cellulose (AVICEL®) per mland 4 mg per g cellulose of a composition containing a blend of anAspergillus aculeatus GH10 xylanase (WO 94/021785) and a Trichodermareesei cellulase preparation containing Aspergillus fumigatusbeta-glucosidase (WO 2005/047499) and Thermoascus aurantiacus GH61Apolypeptide (WO 2005/074656) in 50 mM sodium acetate, 1 mM manganesesulfate at pH 5.0 in the presence of zero or 0.1% (w/w) Kraft (Indulin)lignin or oxidized Kraft (Indulin) lignin. Oxidized Kraft lignin wasgenerated by overnight incubation of a 20% total solids slurry of ligninwith sodium periodate at 4° C. at a concentration of 5 g of sodiumperiodate per 100 g of slurry. The oxidized lignin was washedextensively with water and then freeze dried. The T. reesei cellulasecomposition was either supplemented with an additional 15% (w/w) T.aurantiacus GH61A polypeptide or was not supplemented.

FIG. 26 shows the concentration of glucose produced by saccharificationat various saccharification times as indicated. Addition of lignin oroxidized lignin enhanced the saccharification of AVICEL® by the T.reesei cellulase composition containing GH61A polypeptide. Addition of15% (w/w) supplemental GH61A polypeptide to a T. reesei cellulasecomposition containing T. aurantiacus GH61A polypeptide furtherincreased the extent of saccharification of a hydrolysis reactioncontaining Kraft lignin. Addition of 15% supplemental GH61A polypeptideto a cellulase composition containing GH61A polypeptide resulted inlower glucose conversion than comparable reactions without supplementalGH61A polypeptide in hydrolyses of AVICEL® containing no Kraft lignin orcontaining oxidized Kraft lignin. These data indicate that products ofbiomass pretreatment derived from specific lignins or the ligninsthemselves, in combination with GH61A polypeptide, enhance theconversion of cellulose by a T. reesei cellulase composition.

Example 37 Thermoascus aurantiacus GH61A Enhancement of Cellulolysis ofHigh Total Solids Alkaline Pretreated Corn Stover by a Trichodermareesei Cellulase Composition

Raw, washed and milled corn stover was pretreated with 8% (w/w of dryweight) sodium hydroxide at 90° C. for 1 hour. The resulting wholeslurry was transferred to a vacuum filtration apparatus and washedexhaustively with tap water until the pH of the filtrate was less thanor equal to 8.6. The washed solids were then transferred to hydrolysisreactors to give final solids concentrations of 10%. A compositioncontaining a blend of an Aspergillus aculeatus GH10 xylanase (WO94/021785) and a Trichoderma reesei cellulase preparation containingAspergillus fumigatus beta-glucosidase (WO 2005/047499) and Thermoascusaurantiacus GH61A polypeptide (WO 2005/074656) was replaced withincreasing concentrations of GH61A polypeptide, maintaining a fixedtotal protein concentration of 4 mg protein per gram cellulose. Thewashed, milled, alkaline pretreated corn stover was hydrolyzed at 50° C.for 120 hours in 10-20 g Oak Ridge tubes (Nalge Nunc InternationalCorporation, Rochester, N.Y., USA) containing ¼ inch steel balls foragitation, using a FINEPCR Hybridization Oven (Daigger, Inc. VernonHills, Ill., USA), rotating at 12 rpm.

FIG. 27 shows that glucose concentration increased with increasingreplacement of the cellulase composition with T. aurantiacus GH61Apolypeptide, indicating that cellulose hydrolysis was enhanced with5-10% additional GH61A polypeptide. Thus at high total solids,supplementation of a T. reesei cellulase composition with higherrelative concentrations of GH61 polypeptide resulted in higher celluloseconversion of alkaline pretreated corn stovers.

Example 38 Thermoascus aurantiacus GH61A Enhancement of Cellulolysis ofHigh Total Solids Pretreated Corn Stover of Various PretreatmentSeverities by a Trichoderma reesei Cellulase Composition

Raw corn stover was milled in a Thomas Model 10 Wiley Mill (ThomasScientific, Swedesboro, N.J., USA) with a nominal screen size of 2 mmand then thoroughly washed with tap water. The washed corn stover wasallowed to dry in a 45° C. convection oven until the dry solids contentwas above 90%. The dried solids were then sieved through a #40 meshscreen to ensure a uniform size distribution. An Accelerated SolventExtractor (ASE) 350 instrument (Dionex Corporation, Bannockburn, Ill.,USA) was used for all pretreatments. Approximately 15.0 g of the washed,milled and sieved corn stover was packed into a 100 ml stainless steelextraction cell to ensure consistent solids loading during pretreatment.The extraction cell was loaded into the heating chamber and filled withsulfuric acid solution of various concentrations until the back-pressurereached 1500 psi. The heating chamber then heated the filled cell to thedesired temperature. At the end of the heating phase, the pretreatmentwas stopped by immediately releasing the pressure in the extractioncell, purging with nitrogen gas, and collecting the pretreatment liquorin a designated glass vial. The extraction cell was then immediatelyremoved from the ASE 350 instrument and quenched in ice. After coolingto room temperature, the pretreated solids were removed from the celland re-slurried with the pretreatment liquor. The following pretreatmentconditions were varied: temperature from 150-190° C., static residencetimes between 1 and 15 minutes, and acid concentrations between0.4%-1.0% (w/w) H₂SO₄. The range for combined severity factor rangedfrom 0.5 to 2.0.

Pretreated corn stover at each pretreatment severity was adjusted to pH5.0 and a final TS of 15%. Hydrolysis was initiated by adding to eachPCS batch either 2 mg of a composition containing a blend of anAspergillus aculeatus GH10 xylanase (WO 94/021785) and a Trichodermareesei cellulase preparation containing Aspergillus fumigatusbeta-glucosidase (WO 2005/047499) and Thermoascus aurantiacus GH61Apolypeptide (WO 2005/074656) with 0.4 mg of T. aurantiacus GH61Apolypeptide per gram cellulose or 1.6 mg of the composition containing ablend of an Aspergillus aculeatus GH10 xylanase (WO 94/021785) and aTrichoderma reesei cellulase preparation containing Aspergillusfumigatus beta-glucosidase (WO 2005/047499) and Thermoascus aurantiacusGH61A polypeptide (WO 2005/074656) with 0.4 mg of T. aurantiacus GH61Apolypeptide per gram cellulose and incubating at 50° C. for up to 216hrs in 10-20 g Oak Ridge tubes containing ¼ inch steel balls foragitation, using a FINEPCR Hybridization Oven, rotating at 12 rpm.

FIG. 28 shows the cellulose conversion of high total solids (15% dryweight) corn stover of various severity acid pretreatments. At each setof pretreatment conditions, except for a single, low severitypretreatment, replacement of the cellulase composition with 20%additional T. aurantiacus GH61A polypeptide resulted in greatercellulose conversion, particularly at 216 hours of hydrolysis. Graybars: 120 hours of saccharification; black bars: 216 hours ofsaccharification. Thus at high total solids, supplementation of a T.reesei cellulase composition with higher relative concentrations of GH61polypeptide resulted in higher cellulose conversion when thepretreatment severity was sufficient to generate suitable biomassliquor.

Example 39 Thermoascus aurantiacus GH61A Enhancement of Cellulolysis ofHigh Total Solids Pretreated Giant Cane (Arundo donax) of VariousPretreatment Severities by a Trichoderma reesei Cellulase Composition

Raw Arundo donax was milled in a Thomas Model 10 Wiley Mill with anominal screen size of 2 mm and then thoroughly washed with tap water.The washed A. donax biomass was allowed to dry in a 45° C. convectionoven until the dry solids content was above 90%. The dried solids werethen sieved through a #40 mesh screen to ensure a uniform sizedistribution. An Accelerated Solvent Extractor (ASE) 350 instrument wasused for all pretreatments. Approximately 20.0 g of the washed, milledand sieved Arundo donax was packed into a 100 ml stainless steelextraction cell to ensure consistent solids loading during pretreatment.The extraction cell was loaded into the heating chamber and filled withsulfuric acid solution of various concentrations until the back-pressurereached 1500 psi. The heating chamber then heated the filled cell to thedesired temperature. At the end of the heating phase, the pretreatmentwas stopped by immediately releasing the pressure in the extractioncell, purging with nitrogen gas and collecting the pretreatment liquorin a designated glass vial. The extraction cell was then immediatelyremoved from the ASE 350 instrument and quenched in ice. After coolingto room temperature, the pretreated solids were removed from the celland re-slurried with the pretreatment liquor. The following pretreatmentconditions were varied: temperature from 170-190° C., static residencetimes between 1 and 5 minutes, and acid concentrations between 0.5%-1.0%(w/w) H₂SO₄. The range for combined severity factor ranged from 0.35 to2.13.

Arundo donax biomass of each pretreatment severity was adjusted to pH5.0 and a final TS of 15%. Hydrolysis was initiated by adding either 4mg of a composition containing a blend of an Aspergillus aculeatus GH10xylanase (WO 94/021785) and a Trichoderma reesei cellulase preparationcontaining Aspergillus fumigatus beta-glucosidase (WO 2005/047499) andThermoascus aurantiacus GH61A polypeptide (WO 2005/074656) per gramcellulose or 3.4 mg of the a composition containing a blend of anAspergillus aculeatus GH10 xylanase (WO 94/021785) and a Trichodermareesei cellulase preparation containing Aspergillus fumigatusbeta-glucosidase (WO 2005/047499) and Thermoascus aurantiacus GH61Apolypeptide (WO 2005/074656) and 0.6 mg of T. aurantiacus GH61A per gramcellulose and incubating at 50° C. for up to 120 hours in 10-20 g OakRidge tubes containing ¼ inch steel balls for agitation, using a FINEPCRHybridization Oven, rotating at 12 rpm.

FIG. 29 shows the cellulose conversion of high total solids (15% dryweight) Arundo donax of various severity acid pretreatments. At each setof pretreatment conditions, replacement of the cellulase compositionwith 15% additional T. aurantiacus GH61A polypeptide resulted in greatercellulose conversion, particularly at 216 hours of hydrolysis. Graybars: 72 hours of saccharification; black bars: 120 hours ofsaccharification. Thus at high total solids, supplementation of a T.reesei cellulase composition with higher relative concentrations of GH61polypeptide resulted in higher cellulose conversion of A. donax biomass.

The present invention is further described by the following numberedparagraphs:

[1] A method for degrading or converting a cellulosic material,comprising: treating the cellulosic material with an enzyme compositionin the presence of a polypeptide having cellulolytic enhancing activityand a liquor, wherein the combination of the polypeptide havingcellulolytic enhancing activity and the liquor enhances hydrolysis ofthe cellulosic material by the enzyme composition.

[2] The method of paragraph 1, wherein the cellulosic material ispretreated.

[3] The method of paragraph 1 or 2, further comprising recovering thedegraded cellulosic material.

[4] The method of any of paragraphs 1-3, wherein the enzyme compositioncomprises one or more (several) enzymes selected from the groupconsisting of a cellulase, a hemicellulase, an esterase, an expansin, alaccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease,and a swollenin.

[5] The method of paragraph 4, wherein the cellulase one or more(several) enzymes selected from the group consisting of anendoglucanase, a cellobiohydrolase, and a beta-glucosidase.

[6] The method of paragraph 4, wherein the hemicellulase is one or more(several) enzymes selected from the group consisting of a xylanase, anacetyxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[7] The method of any of paragraphs 1-6, wherein the degraded cellulosicmaterial is a sugar.

[8] The method of paragraph 7, wherein the sugar is selected from thegroup consisting of glucose, xylose, mannose, galactose, and arabinose.

[9] A method for producing a fermentation product, comprising:

(a) saccharifying a cellulosic material with an enzyme composition inthe presence of a polypeptide having cellulolytic enhancing activity anda liquor, wherein the combination of the polypeptide having cellulolyticenhancing activity and the liquor enhances hydrolysis of the cellulosicmaterial by the enzyme composition;

(b) fermenting the saccharified cellulosic material with one or morefermenting microorganisms to produce the fermentation product; and

(c) recovering the fermentation product from the fermentation.

[10] The method of paragraph 9, wherein the cellulosic material ispretreated.

[11] The method of paragraph 9 or 10, wherein the enzyme compositioncomprises one or more (several) enzymes selected from the groupconsisting of a cellulase, a hemicellulase, an expansin, an esterase, alaccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease,and a swollenin.

[12] The method of paragraph 11, wherein the cellulase is one or more(several) enzymes selected from the group consisting of anendoglucanase, a cellobiohydrolase, and a beta-glucosidase.

[13] The method of paragraph 11, wherein the hemicellulase is one ormore (several) enzymes selected from the group consisting of a xylanase,an acetyxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[14] The method of any of paragraphs 9-13, wherein steps (a) and (b) areperformed simultaneously in a simultaneous saccharification andfermentation.

[15] The method of any of paragraphs 9-14, wherein the fermentationproduct is an alcohol, an alkane, a cycloalkane, an alkene, an aminoacid, a gas, isoprene, a ketone, an organic acid, or polyketide.

[16] A method of fermenting a cellulosic material, comprising:fermenting the cellulosic material with one or more fermentingmicroorganisms, wherein the cellulosic material is saccharified with anenzyme composition in the presence of a polypeptide having cellulolyticenhancing activity and a liquor, wherein the combination of thepolypeptide having cellulolytic enhancing activity and the liquorenhances hydrolysis of the cellulosic material by the enzymecomposition.

[17] The method of paragraph 16, wherein the cellulosic material ispretreated before saccharification.

[18] The method of paragraph 16 or 17, wherein the enzyme compositioncomprises one or more (several) enzymes selected from the groupconsisting of a cellulase, a hemicellulase, an expansin, an esterase, alaccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease,and a swollenin.

[19] The method of paragraph 18, wherein the cellulase is one or more(several) enzymes selected from the group consisting of anendoglucanase, a cellobiohydrolase, and a beta-glucosidase.

[20] The method of paragraph 18, wherein the hemicellulase is one ormore (several) enzymes selected from the group consisting of a xylanase,an acetyxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[21] The method of any of paragraphs 16-20, wherein the fermenting ofthe cellulosic material produces a fermentation product.

[22] The method of paragraph 21, further comprising recovering thefermentation product from the fermentation.

[23] The method of any of paragraphs 16-22, wherein the fermentationproduct is an alcohol, an alkane, a cycloalkane, an alkene, an aminoacid, a gas, isoprene, a ketone, an organic acid, or polyketide.

[24] The method of any of paragraphs 1-23, wherein the liquor isobtained from a material that is the same as the cellulosic materialsubjected to saccharification by the enzyme composition.

[25] The method of any of paragraphs 1-23, wherein the liquor isobtained from a material that is different than the cellulosic materialsubjected to saccharification by the enzyme composition.

[26] The method of any of paragraphs 1-23, wherein the liquor isobtained from a material that is the same as the cellulosic materialsubjected to saccharification by the enzyme composition, and thetreatment conditions used to produce the liquor are different from thepretreatment conditions of the cellulosic material.

[27] The method of any of paragraphs 1-23, wherein the liquor isobtained from a material that is the same as the cellulosic materialsubjected to saccharification by a cellulase composition, and thetreatment conditions used to produce the liquor are the same as thepretreatment conditions of the cellulosic material.

[28] The method of any of paragraphs 1-23, wherein the liquor isobtained from a material that is the same as the cellulosic materialsubjected to saccharification by the enzyme composition, and thetreatment conditions used to produce the liquor are the same as thepretreatment conditions of the cellulosic material, and the liquor isfurther processed to remove cellulose inhibitors.

[39] The method of any of paragraphs 1-23, wherein the liquor isobtained from a material that is different than the cellulosic materialsubjected to saccharification by the enzyme composition, and thetreatment conditions used to produce the liquor are different from thepretreatment conditions of the cellulosic material.

[30] The method of any of paragraphs 1-23, wherein the liquor isobtained from a material that is different than the cellulosic materialsubjected to saccharification by the enzyme composition, and thetreatment conditions used to produce the liquor are the same as thepretreatment conditions of the cellulosic material.

[31] The method of any of paragraphs 1-23, wherein the liquor isobtained from a material that is different than the cellulosic materialsubjected to saccharification by the enzyme composition, and thetreatment conditions used to produce the liquor are the same as thepretreatment conditions of the cellulosic material, and the liquor isfurther processed to remove cellulose inhibitors.

[32] The method of any of paragraphs 1-31, wherein the liquor optimizesthe cellulolytic enhancing activity of a GH61 polypeptide with a GH61effect of preferably at least 1.05, more preferably at least 1.10, morepreferably at least 1.15, more prefereably at least 1.2, more preferablyat least 1.25, more preferably at least 1.3, more preferably at least1.35, more preferably at least 1.4, more preferably at least 1.45, morepreferably at least 1.5, more preferably at least 1.55, more preferablyat least 1.6, more preferably at least 1.65, more preferably at least1.7, more preferably at least 1.75, more preferably at least 1.8, morepreferably at least 1.85, most preferably at least 1.9, most preferablyat least 1.95, and even most preferably at least 2.

[33] The method of any of paragraphs 1-31, wherein an effective amountof the liquor to cellulose is about 10⁻⁶ to about 10 g per g ofcellulose, e.g., about 10⁻⁶ to about 7.5 g, about 10⁻⁶ to about 5, about10⁻⁶ to about 2.5 g, about 10⁻⁶ to about 1 g, about 10⁻⁵ to about 1 g,about 10⁻⁵ to about 10⁻¹ g, about 10⁻⁴ to about 10⁻¹ g, about 10⁻³ toabout 10⁻¹ g, or about 10⁻³ to about 10⁻² g per g of cellulose.

[34] The method of any of paragraphs 1-31, wherein the liquor is presentin an amount that minimizes inhibition of a cellulase composition ofabout 1 to about 20% (v/v), e.g., about 1 to about 15%, about 1 to about10%, about 2 to about 7%, about 2 to about 5%, or about 3 to about 5%.

[35] The method of any of paragraphs 1-31, wherein the liquor isobtained from a lignocellulose material, a hemicellulose material, alignacious material, monosaccharides of the lignocellulose material,monosaccharides of the hemicellulose material, or a combination thereof.

[36] The method of any of paragraphs 1-35, wherein the liquor is furtherprocessed to remove inhibitors of a cellulase, a hemicellulase, or acombination thereof.

[37] An isolated liquor, which in combination with a polypeptide havingcellulolytic enhancing activity enhances hydrolysis of a cellulosicmaterial by a cellulolytic enzyme.

[38] A composition comprising a polypeptide having cellulolyticenhancing activity and a liquor, wherein the combination of thepolypeptide having cellulolytic enhancing activity and the liquorenhances hydrolysis of a cellulosic material by a cellulolytic enzyme.

[39] The composition of paragraph 38, which further comprises one ormore (several) enzymes selected from the group consisting of acellulase, a hemicellulase, an esterase, an expansin, a laccase, aligninolytic enzyme, a pectinase, a peroxidase, a protease, and aswollenin.

[40] The composition of paragraph 39, wherein the cellulase one or more(several) enzymes selected from the group consisting of anendoglucanase, a cellobiohydrolase, and a beta-glucosidase.

[41] The composition of paragraph 39, wherein the hemicellulase is oneor more (several) enzymes selected from the group consisting of axylanase, an acetyxylan esterase, a feruloyl esterase, anarabinofuranosidase, a xylosidase, and a glucuronidase.

[42] The composition of any of paragraphs 38-41, wherein the liquoroptimizes the cellulolytic enhancing activity of a GH61 polypeptide witha GH61 effect of preferably at least 1.05, more preferably at least1.10, more preferably at least 1.15, more prefereably at least 1.2, morepreferably at least 1.25, more preferably at least 1.3, more preferablyat least 1.35, more preferably at least 1.4, more preferably at least1.45, more preferably at least 1.5, more preferably at least 1.55, morepreferably at least 1.6, more preferably at least 1.65, more preferablyat least 1.7, more preferably at least 1.75, more preferably at least1.8, more preferably at least 1.85, most preferably at least 1.9, mostpreferably at least 1.95, and even most preferably at least 2.

[43] The composition of any of paragraphs 38-41, wherein an effectiveamount of the liquor to cellulose is about 10⁻⁶ to about 10 g per g ofcellulose, e.g., about 10⁻⁶ to about 7.5 g, about 10⁻⁶ to about 5, about10⁻⁶ to about 2.5 g, about 10⁻⁶ to about 1 g, about 10⁻⁵ to about 1 g,about 10⁻⁵ to about 10⁻¹ g, about 10⁻⁴ to about 10⁻¹ g, about 10⁻³ toabout 10⁻¹ g, or about 10⁻³ to about 10⁻² g per g of cellulose.

[44] The composition of any of paragraphs 38-41, wherein the liquor isobtained from a lignocellulose material, a hemicellulose material, alignacious material, monosaccharides of the lignocellulose material,monosaccharides of the hemicellulose material, or a combination thereof.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

What is claimed is:
 1. A method for producing a fermentation product,comprising: (a) saccharifying a pretreated cellulosic material with anenzyme composition in the presence of a GH61 polypeptide havingcellulolytic enhancing activity and a liquor, wherein the combination ofthe GH61 polypeptide having cellulolytic enhancing activity and theliquor enhances hydrolysis of the pretreated cellulosic material by theenzyme composition compared to the GH61 polypeptide alone or the liquoralone, wherein (i) the liquor is obtained from a material that is thesame as the cellulosic material and the treatment conditions used toproduce the liquor are different from the conditions used to produce thepretreated cellulosic material, or (ii) the liquor is obtained from amaterial that is different than the cellulosic material and thetreatment conditions used to produce the liquor are the same as ordifferent from the conditions used to produce the pretreated cellulosicmaterial; (b) fermenting the saccharified cellulosic material with oneor more fermenting microorganisms to produce the fermentation product;and (c) recovering the fermentation product from the fermentation. 2.The method of claim 1, wherein the liquor is further processed to removeinhibitors of a cellulase, a hemicellulase, or a combination thereof. 3.The method of claim 1, wherein the liquor is obtained from alignocellulose material, a hemicellulose material, a lignaciousmaterial, monosaccharides of the lignocellulose material,monosaccharides of the hemicellulose material, or a combination thereof.4. The method of claim 1, wherein the enzyme composition comprises oneor more enzymes selected from the group consisting of a cellulase, ahemicellulase, an esterase, an expansin, a laccase, a ligninolyticenzyme, a pectinase, a peroxidase, a protease, and a swollenin.
 5. Themethod of claim 4, wherein the cellulase is one or more enzymes selectedfrom the group consisting of an endoglucanase, a cellobiohydrolase, anda beta-glucosidase.
 6. The method of claim 4, wherein the hemicellulaseis one or more enzymes selected from the group consisting of a xylanase,an acetyxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.
 7. The method of claim 1, wherein steps(a) and (b) are performed simultaneously in a simultaneoussaccharification and fermentation.
 8. The method of claim 1, wherein thethe fermentation product is an alcohol, an alkane, a cycloalkane, analkene, an amino acid, a gas, isoprene, a ketone, an organic acid, orpolyketide.
 9. The method of claim 1, wherein the liquor optimizes thecellulolytic enhancing activity of a GH61 polypeptide with a GH61 effectof at least 1.05.
 10. The method of claim 1, wherein an effective amountof the liquor to cellulose is about 10⁻⁶ to about 10 g per g ofcellulose.
 11. The method of claim 1, wherein the liquor is present inan amount that minimizes inhibition of a cellulase composition of about1 to about 20% (v/v).